Inhibition of innate immune response

ABSTRACT

The present invention provides methods, kits, and compositions for reducing an innate immune system response in a human or animal cell, tissue or organism. One embodiment comprises: introducing an Agent mRNA comprising in vitro-synthesized mRNA encoding one or more proteins that affect the induction, activity or response of an innate immune response pathway; whereby, the innate immune response in the cell, tissue or organism is reduced compared to the innate immune response in the absence of the Agent mRNA. Other embodiments are methods, compositions and kits for using an Agent mRNA for treating a disease or medical condition in a human or animal that exhibits symptoms of an elevated innate immune system, or for reducing an innate immune response that is induced in a human or animal cell, tissue or organism by a Foreign Substance that is administered to the cell, tissue or organism.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/129,703, filed Apr. 2, 2014, which is a U.S. § 371 National EntryApplication of International Patent Application PCT/US2012/044418, filedJun. 27, 2012, which claims the priority benefit of U.S. ProvisionalPatent Application No. 61/501,420, each of which are incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention provides methods, kits, and compositions forreducing an innate immune system response in a human or animal cell,tissue or organism by introducing into the cell, tissue or organism anAgent mRNA comprising or consisting of mRNA encoding one or moreproteins that affect the induction, activity and/or response of aninnate immune response pathway; whereby, the innate immune response inthe cell, tissue or organism is reduced compared to the innate immuneresponse in the absence of introducing the Agent mRNA. Other embodimentsare compositions or kits comprising an Agent mRNA for treating a diseaseor medical condition in a human or animal that exhibits symptoms of anelevated innate immune system, or for reducing an innate immune responsethat is induced in a human or animal cell, tissue or organism by aForeign Substance that is administered to the cell, tissue or organismfor a biological, medical, agricultural or research purpose.

BACKGROUND

The innate immune systems of humans and animals comprise a number ofdifferent mechanisms by which cells in these organisms recognize andrespond to a variety of different pathogen-associated molecular patterns(PAMPS) on foreign substances or damage-associated (ordanger-associated) molecular patterns due to components released due todamaged cells or stress signals from damaged cells (DAMPS). Patternrecognition receptor (PRR) proteins, including membrane-bound toll-likereceptors (TLRs) and cytoplasmic NOD-like receptors (NLRs), recognize avariety of different ligands as foreign, damaged, or non-self (e.g.,pathogen-associated or damage-associated) and activate one or moreinnate immune response pathways that function to defend the organism. Insome cases, the innate immune response pathway may result in damage ordeath of the cell after binding of a ligand on a foreign substance tothe PRR of the cell. For example, a type I interferon (IFN) response isinduced upon binding of double-stranded RNA (dsRNA) to toll-likereceptor 3 (TLR3) or binding of a Gram-negative lipopolysaccharide (LPS)to TLR4, and these IFN responses can inhibit protein translation in thecell and induce many other innate immune response pathways that resultin damage to the cell, or, if sustained over time (e.g., by repeatedexposure to the foreign substance comprising dsRNA or LPS over multipledays), result in death of the cell.

The Interferon family of cytokines is one key component of the innateimmune response to both bacterial and viral infection. Interferons werediscovered more than 50 years ago as biological agents that inhibitedthe replication of influenza virus (Isaacs and Lindenmann, 1957).Interferons are designated type I-III based on the receptor complex theysignal through. Type I IFNs, which comprise 13 IFNα subtypes, IFNβ,IFNκ, IFNε, IFNo, IFNτ and IFNδ, engage the ubiquitously expressed IFNAR(IFNα receptor) complex that is composed of the IFNAR1 and IFNAR2subunits. The functions of Type I IFNs are well characterized and knownto be essential for mounting a robust anti-viral response (Muller etal., 1994). Type II IFNs consist of the single IFNγ protein that bindsthe IFNγ receptor (IFNGR) complex. IFNγ secretion functions primarily toinhibit pathogens other than viruses. Type III IFNs consist of 3 IFNλsand signal through IFNLR1 and IL-10R2. At present, not much is knownregarding type III IFNs other than that they are known to regulate anantiviral response and may be the ancestral type I IFNs (Levraud et al.,2007).

Elevated type I IFN levels have been shown to play major roles in thedisease states in autoimmune disorders such as psoriasis and systemiclupus erythematosus (SLE) (Hua et al., 2006; Kirou et al., 2005; Nestleet al., 2005). Neutralization of type I IFNs or type I IFN receptorswith anti-interferon pathway-specific antibodies have been shown toreduce psoriasis and SLE disease progression (Nestle et al., 2005; Yaoet al., 2009).

Viral infections initiate an innate immune response in infected cellsresulting in a cascade of intracellular events, ultimately resulting inthe secretion of interferons. Triggering the innate immune response canresult in apoptosis of the cell or inhibition or repression of proteinsynthesis. Immunorecognition of viruses is dependent on detection ofviral nucleic acids by PPRs, including TLRs. TLR3 activates an innateimmune response by recognizing and binding to virally-derived dsRNA(Alexopoulou et al., 2001; Wang et al., 2004). TLR9 is activated by DNAcontaining unmethylated CpG motifs, found in viral and bacterial DNA(Hemmi et al., 2000). Single-stranded RNAs (ssRNA) and small interferingRNA (siRNAs) are recognized by TLR7 and TLR8 (Diebold et al., 2004; Heilet al., 2004; Hemmi et al., 2002; Judge and MacLachlan, 2008). TLR4activates an innate immune response by recognizing and binding to LPS ofGram-negative bacteria. Innate immune responses induced by differentforeign substances activating different TLRs can be mediated, at leastin part, through common signaling pathways. For example, activation ofboth TLR3 and TLR4 trigger signaling pathways that result in productionof type I interferons (IFNs).

Vaccinia virus (VV), a cytoplasmic DNA virus in the poxvirus family,encodes a set of intracellular proteins or soluble cytokine bindingproteins that enhance virus virulence. VV intracellular E3L protein, aninhibitor of interferon induction, binds to dsRNA and prevents theactivation of the IFN-induced protein kinase PKR (Chang et al., 1992).VV intracellular K3L protein binds competitively to PKR and blocks thephosphorylation and inactivation of host eIF-2α (Beattie et al., 1991).VV also encodes a secreted IFNα/β receptor that is encoded by the B18Rgene (Colamonici et al., 1995; Symons et al., 1995). This B18R geneencodes a secreted glycoprotein that binds to and inhibits the functionof type I interferons (IFNα/β), while not binding nor inhibiting type IIinterferons (IFNγ) (Symons et al., 1995). Vaccinia strains lackingfunctional B18R show much lower levels of viral virulence demonstratingthe importance of inhibiting type I interferons during viral infection(Colamonici et al., 1995; Symons et al., 1995).

In vitro-transcribed mRNA made from SP6, T7 or T3 RNA polymerases havebeen shown to function in countless studies when used for directinjection into Xenopus laevis (frog) or Danio rerio (zebrafish) ooyctesas well as for transfection into mammalian cells in culture. It is wellestablished that in vitro transcription using T7 RNA polymerase canresult in the generation of some dsRNA in addition to the desired ssRNA(Cazenave and Uhlenbeck, 1994; Triana-Alonso et al., 1995). Introductionof viral dsRNA or the synthetic dsRNA cohomopolymerpolyinosinic-polycytidylic acid (polyI:C) results in the activation of aTLR3-mediated innate immune response (Alexopoulou et al., 2001; Schulzet al., 2005). Similarly, introduction of in vitro-transcribed mRNA ordsRNA into mammalian cells results in the activation of TLR3-mediatedinnate immune response, signified by the production of type Iinterferons (Kariko et al., 2004). Addition of recombinant B18R proteinto the media of cells transfected with in vitro-transcribed mRNAsreduces mRNA-induced toxicity, presumably through the inhibition ofinterferon activity (Angel and Yanik, 2010; Warren et al., 2010); andU.S. Patent Application No. 20100273220)

SUMMARY OF THE INVENTION

The present invention comprises methods, kits, systems, and compositionsfor reducing, suppressing or preventing an innate immune system responsethat is induced in a human or animal cell, tissue or organism. Oneembodiment is a method for reducing, suppressing and/or preventing aninnate immune response in a human or animal cell, tissue or organism,comprising: introducing into the cell, tissue or organism an Agent mRNAcomprising or consisting of mRNA (e.g., in vitro synthesized mRNA)encoding one or more proteins that affect the induction, activity orresponse of an innate immune response pathway; whereby, the innateimmune response in the cell, tissue or organism is reduced, suppressedor prevented compared to the innate immune response in the absence ofintroducing the Agent mRNA. Other embodiments are compositions or kitscomprising an Agent mRNA.

In some embodiments of the methods, compositions and kits, the AgentmRNA comprises or consists of one or more mRNAs encoding one or moreproteins that reduces the activity an innate immune response. In someembodiments, Agent mRNA encodes a protein that binds a biochemicalmolecule (e.g., a protein) in a cell that mediates said innate immuneresponse, which binding reduces the innate immune response. In someembodiments, Agent mRNA encodes an antibody or artificial antibody thatbinds a biochemical molecule (e.g., a protein) that mediates said innateimmune response in a cell, which binding reduces the innate immuneresponse.

In some embodiments of the methods, compositions and kits, the AgentmRNA comprises or consists of one or more mRNAs encoding one or moreproteins that inhibits the activity of an innate immune effector proteinin a signaling pathway mediated by a TLR selected from the groupconsisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 andTLR10. In some embodiments, the Agent mRNA encodes a biologically activefragment, analog or variant of any of said proteins.

In some embodiments of the methods, compositions and kits, the AgentmRNA comprises or consists of one or more mRNAs encoding a biologicallyinactive fragment, mutant, analog or variant or a dominant negativefunctional inhibitor of one or more proteins selected from the groupconsisting of: TP53, TLR3, TLR4, TLR7, TLR8, RARRES3, IFNA1, IFNA2,IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16,IFNA17, IFNA21, IFNK, IFNB1, IL6, TICAM1, TICAM2, MAVS, STAT1, STAT2,EIF2AK2, IRF3, TBK1, CDKN1A, CDKN2A, RNASEL, IFNAR1, IFNAR2, OAS1, OAS2,OAS3, OASL, RB1, ISG15, MX1, IRF9, ISG20, IFIT1, IFIT2, IFIT3, IFIT5,PKR, RIG-1, MDA5, NF-κB, TRIF, Tyk2, and IRF7.

As used herein, a “biologically inactive fragment, mutant, analog orvariant of a protein” or a “dominant negative inhibitor” means afragment, mutant, analog or variant of a wild-type protein thatinteracts with the same cellular molecules as the biologically activewild-type protein, but which, due to a lack of certain amino acids ormoieties in said biologically inactive, fragment, mutant, analog orvariant (of a) protein compared to said wild-type protein, saidinteraction with said biologically inactive fragment, mutant, analog orvariant protein is not biologically active and blocks some aspect of thenormal biological function compared to the interaction with thewild-type protein.

In some embodiments of the methods, compositions and kits, the AgentmRNA comprises or consists of mRNA encoding one or more proteininhibitors (e.g., one or more antibodies or artificial antibodies) thatinhibit the functions of one or more proteins selected from the groupconsisting of: TP53, TLR3, TLR4, TLR7, TLR8, RARRES3, IFNA1, IFNA2,IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16,IFNA17, IFNA21, IFNK, IFNB1, IL6, TICAM1, TICAM2, MAVS, STAT1, STAT2,EIF2AK2, IRF3, TBK1, CDKN1A, CDKN2A, RNASEL, IFNAR1, IFNAR2, OAS1, OAS2,OAS3, OASL, RB1, ISG15, MX1, IRF9, ISG20, IFIT1, IFIT2, IFIT3, IFIT5,PKR, RIG-1, MDA5, NF-κB, TRIF, Tyk2, and IRF7.

In some embodiments of the methods, compositions and kits, the AgentmRNA comprises or consists of one or more mRNAs encoding one or moreproteins that is a regulator or inhibitor of type I-interferonsignaling, induction, or response.

In some embodiments, Agent mRNA encodes an antibody or artificialantibody that binds a biochemical molecule (e.g., a protein) thatmediates a type I-mediated innate immune response in a cell, whichbinding reduces the type I-mediated innate immune response.

In some embodiments of the methods, compositions and kits, the AgentmRNA comprises or consists of mRNA encoding one or more proteininhibitors (e.g., one or more antibodies or artificial antibodies) thatinhibits type I-interferon signaling, induction, or response.

In some embodiments of the methods, compositions and kits, the AgentmRNA comprises or consists of one or more mRNAs encoding one or moreproteins selected from the group consisting of: Vaccinia virus B18Rprotein, Vaccinia virus E3L protein, Vaccinia virus K3L protein,Influenza A virus NS1 protein, human papilloma virus 18 protein E6,human interferon alpha/beta binding proteins, soluble forms of the humaninterferon alpha receptors, INFAR1, 1INFAR2, or a biologically activefragment, analog or variant of any of said proteins.

In some embodiments, the methods, compositions or kits are used to treata disease or medical condition in a human or animal that exhibits anelevated innate immune system response. In some embodiments, themethods, compositions or kits are used to treat a disease or medicalcondition in a human or animal that exhibits an elevated innate immunesystem response comprising or consisting of an elevated type IIFN-mediated innate immune response (e.g., as exhibited by an elevatedlevel of type I IFN production or response). In some of theseembodiments of methods, kits, systems or compositions, the Agent mRNAencodes one or more proteins that reduce or suppress an innate immuneresponse comprising an elevated level of type I IFN production orresponse. For example, in certain embodiments, the Agent mRNA encodingone or more proteins that reduce or suppress an innate immune responsecomprising an elevated level of type I IFN production or response isselected from the group consisting of B18R protein, E3L protein, K3Lprotein or a biologically active fragment, analog or variant of any ofsaid proteins.

In some embodiments, the Agent mRNA encoding innate immune inhibitorsresults in reduction of elevated type I interferon production indiseases like psoriasis or systemic lupus erythematosus (SLE). Infurther embodiments, the Agent mRNA (e.g., encoding one or more proteinsthat reduces or suppresses type I IFN production or response, such asB18R protein, E3L protein, and K3L protein is administered systemically(with or without complexing of Agent mRNA to a transfection reagent)e.g., by vascular injection; in a liquid or cream delivered topically tothe skin; as an aerosol delivered into the lungs; or by electroporationor injection directly into a tissue, such as heart, liver, muscle,brain, pancreas tissue or any other organ or tissue to reduce elevatedtype I IFN production in diseases like psoriasis, SLE or other diseasescaused by upregulation of type I interferon production, includingdiseases for which the cause has not yet been identified andcharacterized. In certain preferred embodiments, the methods, kits andcompositions results in reduction of elevated type I interferonproduction for treatment of psoriasis or SLE.

In other embodiments, the methods, composition and kits find utility forreducing, suppressing or preventing an innate immune response that isinduced in a human or animal cell, tissue or organism by a ForeignSubstance that is administered to the cell, tissue or organism for abiological, medical, agricultural or research purpose. Thus, in someembodiments of the methods, said innate immune response that is reduced,suppressed or prevented is caused by introduction of a Foreign Substancethat is capable of causing an innate immune response in said cell,tissue or organism by affecting the induction, activity or response ofan innate immune response pathway in said cell, tissue or organism.

As used herein, a “Foreign Substance” means any molecule or ligandagainst which a cell, tissue or organism responds by initiating aninnate immune response involving one or more innate immune responsepathways. For example and without limitation, said molecule or ligandcan be recognized through binding to a PRR, such as a TLR or NLR, asbeing foreign, non-self, a PAMP or a DAMP. Furthermore, for example, butwithout limitation, certain Gram-negative bacterial lipopolysaccharidesare Foreign Substances because, once they are recognized by binding toTLR4 on a cell, an innate immune response comprising a type I interferonresponse is induced. Similarly, certain dsRNA (e.g., double stranded RNAlonger than about 25 bases) is a Foreign Substance because it binds toTLR3 on a cell, which in turn results in induction of an innate immuneresponse comprising a type I interferon response.

One embodiment of the invention is a method for reducing, suppressing orpreventing an innate immune response in a human or animal cell, tissueor organism generated by a Foreign Substance, comprising: introducing tothe cell, tissue or organism an effective amount of Agent mRNA encodingone or more proteins that affect the induction, activity or response ofan innate immune response pathway; whereby, the innate immune responsein the cell, tissue or organism is reduced, suppressed or preventedcompared to the innate immune response in the absence of introducing theAgent mRNA. In certain embodiments of the method, the Foreign Substanceis Exogenous RNA.

In certain embodiments, the Exogenous RNA is Exogenous mRNA. In certainembodiments, introduction of Agent mRNA results in an increase intranslation of the Exogenous mRNA in the human or animal cell and/or adecrease in toxicity (resulting in increased cell survival) of theExogenous mRNA to the cell, tissue or organism compared to the toxicity(or cell survival) in the absence of the Agent mRNA.

In certain embodiments, the Foreign Substance is Exogenous siRNA orExogenous miRNA. In certain embodiments, introduction of Agent mRNAresults in an increase in the specificity (e.g., a decrease in theoff-target Exogenous siRNA- or Exogenous miRNA-mediated decrease inexpression compared to the same results in the absence of the AgentmRNA). In certain embodiments, introduction of Agent mRNA results in adecrease in toxicity (or increase in cell survival) or a decrease intranslational inhibition induced by introducing Exogenous siRNA orExogenous miRNA to the cell, tissue or organism compared to the toxicity(or cell survival) or translational inhibition in the absence of theAgent mRNA.

In certain embodiments, the method results in a biological effect in thecells, tissue or organism. In certain embodiments, the Agent mRNAencodes one or more proteins that reduce the innate immune responseinduced by contacting the cell with a Foreign Substance (e.g., an LPS,(e.g., comprising or consisting of a Gram-negative bacterial LPS), adouble stranded RNA (dsRNA), or Exogenous RNA).

In certain embodiments, the Agent mRNA encodes one or more proteins thatreduces the innate immune response outside of the cell (i.e.,extracellularly); e.g., wherein the protein encoded by the Agent mRNA isa secreted protein, such as B18R protein.

In certain embodiments, the Agent mRNA encodes one or more proteins thatreduce the innate immune response inside of the cell (i.e.,intracellularly); e.g., wherein the protein encoded by the mRNA remainswithin the cell, such as E3L or K3L proteins. Those with knowledge inthe field will understand that it is very difficult to reliably deliveran intracellular protein into a cell efficiently and in active form. Thepresent method of reducing, suppressing or preventing an innate immuneresponse by introducing to the cells, tissues or organism, an Agent mRNAencoding such intracellular proteins provides important advantages andbenefits over all methods previously known in the art. In certainembodiments, the Agent mRNA encodes a protein that reduces or suppressesthe type I interferon-induced cellular toxicity or translationinhibition resulting from the cellular transfection or introduction of aForeign Substance comprising or consisting of Exogenous mRNA. In certainembodiments, the Agent mRNA encodes a protein that reduces the type Iinterferon-induced cellular toxicity or translation inhibition resultingfrom contacting the cell with another Foreign Substance (e.g., aGram-negative bacterial LPS or a viral dsRNA).

One embodiment of the invention is a method for reducing, suppressing orpreventing an innate immune response in a human or animal cell, tissueor organism generated by a Foreign Substance, comprising: introducing tothe cell, tissue or organism an effective amount of Agent mRNA encodingone or more proteins that affect the induction, activity or response ofan innate immune response pathway; whereby, the innate immune responsein the cell, tissue or organism is reduced, suppressed or preventedcompared to the innate immune response in the absence of introducing theAgent mRNA. In certain embodiments, the present invention is a methodfor reducing, suppressing or preventing the innate immune response of ahuman or animal cell, tissue or organism caused by transfection of thecell with Exogenous RNA (e.g. Exogenous mRNA) or with Exogenous siRNA orExogenous miRNA, comprising: introducing to the cell (e.g., a cellculture medium or in a in a tissue or organism) an effective amount ofan Agent mRNA encoding one or more proteins that affect the induction,activity or response of an innate immune response pathway; whereby, theinnate immune response in the cell is reduced, suppressed or preventedcompared to the innate immune response in the absence of introducing theAgent mRNA (i.e., the innate immune response following transfection ofthe cell with the Exogenous RNA (e.g. Exogenous mRNA) or with ExogenoussiRNA or Exogenous miRNA is reduced, suppressed or prevented compared tothe innate immune response following transfection of the cell with theExogenous RNA (e.g. Exogenous mRNA) or with Exogenous siRNA or ExogenousmiRNA without the introduction of the Agent mRNA). In certainembodiments, the Exogenous RNA comprises or consists of Exogenous mRNAthat results in a biological effect in the cell. In some preferredembodiments, said introducing into the cell of the Agent mRNA results ina higher rate of cell survival (i.e., a lower rate of cell death) (e.g.,a 25% . . . 50% . . . 100% . . . or >100% higher rate of cell survival)following transfection of the cell with the Exogenous RNA compared tothe rate of cell survival in the absence of introducing to the cell ofthe Agent mRNA. In some preferred embodiments, the Exogenous RNA isExogenous mRNA and said introducing into the cell of the Agent mRNAresults in a higher translation of said Exogenous mRNA into proteinfollowing said transfection of the cell compared to the level oftranslation in the absence of introducing to the cell of the Agent mRNA(e.g., a 10% . . . 25% . . . 50% . . . 75% . . . 100% . . . or >100%higher level of translation in a cell compared to the level oftranslation in a cell transfected with the same amount of Exogenous mRNAbut without the Agent mRNA).

In certain embodiments of any of the methods comprising transfection ofa Foreign Substance comprising Exogenous siRNA or Exogenous miRNA intothe cell, tissue or organism, the Foreign Substance comprising ExogenoussiRNA or Exogenous miRNA results in a biological effect in the cell,tissue or organism. In some preferred embodiments, introducing of AgentmRNA into the cell, tissue or organism into which the Foreign Substancecomprising Exogenous siRNA or Exogenous miRNA is transfected results ina higher level of specificity or a lower level of off-target effects. Incertain embodiments, introduction of Agent mRNA results in a decrease intoxicity (or increase in cell survival) or a decrease in translationalinhibition induced by said transfection of the Foreign Substancecomprising Exogenous siRNA or Exogenous miRNA to the cell, tissue ororganism compared to the toxicity (or cell survival) or translationalinhibition in the absence of the Agent mRNA. In some preferredembodiments, said transfection of said Exogenous RNA (e.g., ExogenousmRNA) or said Exogenous siRNA or Exogenous miRNA comprises or consistsof multiple sequential transfections of said cell, tissue or organismwith said Exogenous RNA (e.g., Exogenous mRNA) or said Exogenous siRNAor Exogenous miRNA (e.g., multiple transfections at daily, 2-day, 3-day,4-day, 5-day, 6-day, weekly, or monthly intervals or any other intervalsor combination of intervals that is found to be effective for aparticular purpose). In some preferred embodiments, said introducinginto the cell of the Agent mRNA is made together with or atapproximately the same time as said transfection of the cell with saidExogenous RNA. In some embodiments, said introducing into the cell ofthe Agent mRNA is made prior to said transfection of the cell with saidExogenous RNA. In certain embodiments, in addition to introducing saidAgent mRNA, the method further comprises introducing to the cell theprotein encoded by said Agent mRNA.

In some preferred embodiments, said translation of said Exogenous mRNAinto protein following said transfection of the cell results in abiological effect. In some preferred embodiments, said biological effectcomprises or consists of: reprogramming of said cell. In someembodiments, said reprogramming comprises or consists of: (i) inductionof a differentiated cell into a pluripotent stem cell (or “inducedpluripotent stem cell” or “iPS cell” or “iPSC”); (ii) differentiation ofan embryonic stem cell (“eSC”) or iPSC into a cell that exhibits a morehighly specialized state of differentiation; or (iii)transdifferentiation of a cell from one state of differentiation to asecond state of differentiation.

In some other preferred embodiments, said biological effect comprises orconsists of: translation of a protein that is defective or lacking in acell of a human or animal patient that has an error of metabolism (e.g.,due to an inherited genetic disease or a de novo mutation that resultsin a missing or defective gene product, including a missing or defectivegene product comprising or consisting of mRNA and/or protein). Thus, insome embodiments of the methods, kits and compositions, the ForeignSubstance is Exogenous RNA (e.g., Exogenous mRNA) and said ExogenousmRNA encodes the protein that is defective or lacking in said cell,tissue or organism (e.g., due to the inherited genetic disease or a denovo mutation that results in a missing or defective gene product).

In some other preferred embodiments of the method, said cell into whichthe Exogenous mRNA is transfected is an antigen-presenting cell or “APC”(e.g., a dendritic cell, a macrophage, a Langerhans cell, a Kuppfer celland an artificial APC) and said Exogenous mRNA that is transfectedcomprises or consists of one or multiple mRNAs derived from a cancercell from a human or animal patient (e.g., wherein said mRNAs are madeby in vitro transcription (IVT) of cDNA generated from substantially allof the mRNA isolated from one or more cancer cells; e.g., wherein theIVT is part of a method comprising amplification of sense RNA; e.g. asdescribed in U.S. Pat. No. 8,039,214); in some preferred embodiments ofthis method, the cell that is transfected with the Exogenous mRNA isused for immunotherapy of a patient that exhibits the cancer; in someembodiments, the Exogenous mRNA that is used for transfection of the APCis derived or prepared from a cancer cell from the patient and the APCthat is transfected is derived or prepared from the same patient withthe cancer; whereas in some other embodiments, the Exogenous mRNA thatis used for transfection of the APC is derived or prepared from a cancercell from a different patient or is genetically engineered or chemicallysynthesized based on knowledge of one or more known gene products thatare expressed in a cancer cell of the type from which the patientsuffers (e.g., preferably, wherein the one or more gene productsexpressed in the cancer cell are not expressed or are expressed at amuch lower level in a cell of the same type but without the cancer); andin some embodiments the APC is derived or prepared from another human oranimal, including from a cultured human or animal cell, including froman ex vivo-differentiated cell. In some preferred embodiments of themethod, said transfection of said cell (e.g., an APC) with saidExogenous RNA (e.g., Exogenous mRNA) comprises or consists oftransfection of said cell in vivo in a human or animal (e.g., followingintradermal, subdermal, or internodal injection).

In further embodiments of any of the methods comprising introducing intothe cell an effective amount of an Agent mRNA encoding a protein thatreduces or suppresses an innate immune response, prior to introducing tothe cell said Agent mRNA, the method further comprises the step of:contacting the cell with a protein that effectively reduces the innateimmune response due to the Agent mRNA itself; this embodiment preventsor reduces the innate immune response from said introducing of saidAgent mRNA encoding a protein that reduces or suppresses an innateimmune response until such time as said Agent mRNA is active (e.g.,until such time as said Agent mRNA that encodes said protein isexpressed in said cell). In certain embodiments of the method, whereinthe Agent mRNA encodes a protein that reduces or suppresses an innateimmune response by extracellular binding (e.g., Agent mRNA encodingVaccinia virus B18R protein or a biologically active fragment, analog orvariant thereof), the method further comprises contacting the cell withthe protein encoded by said Agent mRNA that encodes the extracellularprotein prior to said introducing into the cell the Agent mRNA. In somepreferred embodiments, the agent mRNA encodes the Vaccinia virus B18Rprotein or a biologically active fragment, analog or variant thereof andthe protein that effectively reduces the innate immune response due tothe Agent mRNA is the Vaccinia virus B18R protein or a biologicallyactive fragment, analog or variant thereof. In other embodiments, saidprotein that effectively reduces the innate immune response due to theAgent mRNA is one or more other proteins that reduces a type 1interferon response in said cell.

In additional embodiments, the present invention is a kit or systemcomprising or consisting of: a) an Agent mRNA that reduces or suppressesthe innate immune response in a cell that is induced by a ForeignSubstance (e.g., a Foreign Substance comprising or consisting of a LPS,dsRNA, Exogenous RNA, or Exogenous siRNA or Exogenous miRNA), and b) anExogenous RNA (e.g., an Exogenous mRNA) or Exogenous siRNA or ExogenousmiRNA. In certain embodiments, the Agent mRNA encodes a protein thatreduces or suppresses the innate immune response induced in the cell bythe Foreign Substance (e.g., induced by transfection with the ExogenousRNA or Exogenous siRNA or Exogenous miRNA). In certain embodiments, thekit or system further comprises the protein encoded by the Agent mRNA.In certain embodiments of a composition or a kit comprising an AgentmRNA encoding a protein that reduces and/or suppresses an innate immuneresponse by intracellular binding or action (e.g., E3L or K3L mRNA), thecomposition or kit further comprises a protein that reduces orsuppresses an innate immune response by extracellular binding or afterbeing secreted from the cell (e.g., B18R protein). In certainembodiments of a composition or a kit comprising an Agent mRNA encodinga protein that reduces or suppresses an innate immune response byextracellular binding or after being secreted from the cell (e.g., AgentmRNA encoding B18R protein), the composition or kit further comprises aprotein that reduces or suppresses an innate immune response encoded bysaid Agent mRNA or another protein that reduces or suppresses an innateimmune response by extracellular binding or after being secreted fromthe cell. In certain embodiments, the kit further comprises the cell.

In some embodiments, the present invention provides a compositioncomprising or consisting of an Agent mRNA that reduces an innate immuneresponse after being introduced into a cell, tissue or organism. In someembodiments, the present invention provides a composition comprising orconsisting of: a) an Agent mRNA that reduces the innate immune responsein a cell, tissue or organism that is induced by transfection withExogenous RNA (e.g., Exogenous mRNA) or Exogenous siRNA or ExogenousmiRNA, and b) the Exogenous RNA or Exogenous siRNA or Exogenous miRNA.In certain embodiments, the composition further comprises the cell.

In particular embodiments of methods, kits or compositions, the AgentmRNA encodes a protein that reduces the biological activity of a proteinin an innate immune response pathway. In some embodiments, the proteinencoded by said Agent mRNA is a viral-encoded protein. In someembodiments of methods, kits or compositions, the Agent mRNA furthercomprises a small molecule that reduces the biological activity of aprotein in an innate immune response pathway. In further embodiments,the methods, compositions, or kits use or comprise an Agent mRNA thatcomprises or consists of two or more different RNAs (e.g., two or moremRNAs encoding two or more different proteins). In some embodiments, theproteins that effectively reduce or suppress the innate immune encodedby Agent mRNA comprise one or more other proteins that reduces a type 1interferon response in the cell, tissue or organism. In some preferredembodiments, the Agent mRNA encodes the Vaccinia virus B18R protein or abiologically active fragment, analog or variant thereof, and the proteineffectively reduces the innate immune response due to the Agent mRNA isthe Vaccinia virus B18R protein or a biologically active fragment,analog or variant thereof.

In certain embodiments of any of the methods, kits systems orcompositions, the Agent mRNA encodes one, two or more proteins selectedfrom the group consisting of B18R protein, E3L protein, K3L protein, ora biologically active fragment, analog or variant of any thereof. Inother embodiments, the methods, kits, systems and compositions hereinemploy two or more of B18R protein, E3L protein, K3L protein (orbiologically active fragments thereof).

In certain embodiments, the Agent mRNA encodes a protein inhibitor oftype I-interferon signaling, induction, or response. In furtherembodiments, the Agent mRNA molecule that encodes a protein inhibitor isselected from the group consisting of: Vaccinia virus B18R protein,human interferon alpha/beta binding proteins, soluble forms of the humaninterferon alpha receptors (e.g., IFNARs; see, U.S. Pat. No. 6,458,932;European Patent No. EP0679717 B1), including INFAR1 and 1INFAR2, or abiologically active fragment, analog or variant of any thereof. Infurther embodiments, the Agent mRNA encodes Vaccinia virus B18R protein,Vaccinia virus E3L protein (an inhibitor of interferon induction),Vaccinia virus K3L protein (an inhibitor of PKR, which is an effectorprotein activated by interferon signaling), Influenza A virus NS1protein (an inhibitor of interferon induction), Human papilloma virus 18protein E6 (an interferron signaling inhibitor), human interferonalpha/beta binding proteins, soluble forms of the human interferon alphareceptors, including INFAR1 and 1INFAR2, or a biologically activefragment, analog or variant of any thereof. In additional embodiments,the Agent mRNA comprises or consists of one or more mRNAs that encodes abiologically inactive fragment, mutant, analog or variant or a dominantnegative functional inhibitor of one or more positive effector proteinsin an innate immune response pathway, wherein said biologically inactivefragment, mutant, analog or variant is of a protein selected from thegroup consisting of: TP53, TLR3, TLR4, TLR7, TLR8, RARRES3, IFNA1,IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14,IFNA16, IFNA17, IFNA21, IFNK, IFNB1, IL6, TICAM1, TICAM2, MAVS, STAT1,STAT2, EIF2AK2, IRF3, TBK1, CDKN1A, CDKN2A, RNASEL, IFNAR1, IFNAR2,OAS1, OAS2, OAS3, OASL, RB1, ISG15, MX1, IRF9, ISG20, IFIT1, IFIT2,IFIT3, IFIT5, PKR, RIG-1, MDA5, NF-κB, TRIF, Tyk2 and IRF7. Inadditional embodiments of the methods, compositions and kits, the AgentmRNA comprises or consists of one or more mRNAs that encodes a proteinthat is a receptor in an innate immune response signaling pathwaymediated by a TLR. In some embodiments of the methods, compositions andkits, in addition to the Agent mRNA, the composition or kit or systemfurther comprises a biologically inactive fragment, mutant, analog orvariant or a dominant negative functional inhibitor of one or morepositive effector proteins selected from the group consisting of: TP53,TLR3, TLR4, TLR7, TLR8, RARRES3, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6,IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21, IFNK,IFNB1, IL6, TICAM1, TICAM2, MAVS, STAT1, STAT2, EIF2AK2, IRF3, TBK1,CDKN1A, CDKN2A, RNASEL, IFNAR1, IFNAR2, OAS1, OAS2, OAS3, OASL, RB1,ISG15, MX1, IRF9, ISG20, IFIT1, IFIT2, IFIT3, IFIT5, PKR, RIG-1, MDA5,NF-κB, TRIF, Tyk2 and IRF7. In still other embodiments, the methods,compositions systems, and kits, further comprises a biologicallyinactive fragment, mutant, analog or variant or a dominant negativefunctional inhibitor of one or more positive effector proteins selectedfrom the group consisting of: TP53, TLR3, TLR4, TLR7, TLR8, RARRES3,IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14,IFNA16, IFNA17, IFNA21, IFNK, IFNB1, IL6, TICAM1, TICAM2, MAVS, STAT1,STAT2, EIF2AK2, IRF3, TBK1, CDKN1A, CDKN2A, RNASEL, IFNAR1, IFNAR2,OAS1, OAS2, OAS3, OASL, RB1, ISG15, MX1, IRF9, ISG20, IFIT1, IFIT2,IFIT3, IFIT5, PKR, RIG-1, MDA5, NF-κB, TRIF, Tyk2 and IRF7. Inadditional embodiments of the methods, compositions and kits, the AgentmRNA encodes a protein that is a biologically active soluble receptor inan innate immune response signaling pathway, or a biologically activefragment, analog or variant thereof. In additional embodiments of themethods, compositions and kits, the Agent mRNA encodes a protein that isan inhibitor that binds a protein in a signaling pathway mediated by aTLR selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9 and TLR10. In some embodiments of the methods,compositions and kits, the Agent mRNA encodes a an antibody orartificial antibody that reduces an innate immune response by binding aprotein in a signaling pathway mediated by a TLR selected from the groupconsisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 andTLR10.

In additional embodiments of the methods, compositions, systems, andkits, the Agent mRNA encodes one or more function-blocking antibodiesthat reduces or suppresses the activity of a protein in an innate immuneresponse pathway. In some embodiments, the sequence of an mRNA encodingan antibody that reduces an innate immune response is first made in anon-human species and then, using any of the methods known in the art,the Agent mRNA in made by modifying the sequence so that the proteinencoded by said Agent mRNA is similar to an antibody which would beproduced naturally in humans; the antibody encoded by said Agent mRNA isthen said to be “humanized” because it is has been adapted to besuitable for use in humans with minimal chance of inducing an activeimmune response. Agent mRNA encoding antibodies intended for use inother species can be similarly adapted for use in those species.

In some embodiments of the methods, kits, systems, and compositions, thecell is a human or animal cell comprising said cell, tissue or organism,selected from the group consisting of: a fibroblast cell, such as fetaland neonatal fibroblasts or adult fibroblasts, an hematopoietic cell, aB cell, a T cell, an APC, including a dendritic cell, a macrophage cell,a Langerhans cell, or an artificial APC, a Kuppfer cell, a monocyte,mononuclear cells, a keratinocyte cell, in particular a primarykeratinocyte, more preferably a keratinocyte derived from hair, anadipose cell, an epithelial cell, an epidermal cell, a chondrocyte, acumulus cell, a neural cell, a glial cell, an astrocyte, a cardiac cell,an esophageal cell, a muscle cell, a melanocyte, and an osteocyte.

In additional embodiments of the methods, kits and compositions, theExogenous mRNA encodes a secreted protein, a cell surface receptor,intracellular signaling mediator or a transcription factor. Inparticular embodiments, the Exogenous mRNA encodes a protein in thefamily selected from the group consisting of OCT3/4, SOX2, KLF4, c-MYC,c-MYC(T58A), L-MYC, NANOG, LIN28, SV40 Large-T antigen, hTERT,E-Cadherin, and MYOD1, SHH, GLI1, RARγ, LRH1, GLIS1, NURR1, MASH1,LMX1A, BRN2, MYT1L, GATA4, MEF2C, TBX5, HAND2, FOXA1, FOXA2, FOXA3,HNF1α, HNF4α, PAX3, and PAX7. In further embodiments, the introducing tothe cell of an effective amount of an Agent mRNA encoding a protein thatreduces or suppresses an innate immune response enhances mRNA-mediatediPS cell generation from somatic cells by repeated transfections withExogenous mRNA encoding one or more proteins selected from the groupconsisting of KLF4, LIN28, c-MYC, L-MYC, c-MYC(T58A), OCT3/4, SOX2,NANOG, GLIS1, RARγ, LRH1, and E-CADHERIN.

In other embodiments, the introducing to the cell of an effective amountof an Agent mRNA encoding a protein that reduces or suppresses theinnate immune response enhances transdifferentiation of one cell typeinto a second cell type. In further embodiments, the introducing to thecell of an effective amount of an Agent mRNA encoding a protein thatreduces the innate immune response enhances differentiation of iPSCs,embryonic stem cells, or lineage-restricted stem cells (e.g.,mesenchymal, hematopoietic, or neuronal stem cells) with Exogenous mRNAencoding factors known to direct stem cells toward various specificdownstream lineages or cell types.

In some embodiments of the methods, kits and compositions, the ForeignSubstance is Exogenous mRNA encoding one or more proteins selected fromthe group consisting of a secreted protein, a cell surface receptor, anintracellular signaling mediator, and a transcription factor,particularly wherein, said transcription factor is a transcriptionfactor protein in the family selected from the group consisting of:OCT3/4, SOX2, KLF4, c-MYC, c-MYC(T58A), L-MYC, NANOG, LIN28, SV40Large-T antigen, hTERT, E-Cadherin, MYOD1, SHH, GLI1, RARγ, LRH1, GLIS1,NURR1, MASH1, LMX1A, BRN2, MYT1L, GATA4, MEF2C, TBX5, HAND2, FOXA1,FOXA2, FOXA3, HNF1α, HNF4α, PAX3 and PAX7. In some preferredembodiments, wherein said Exogenous mRNA encodes OCT3/4, SOX2, KLF4,NANOG, LIN28, and at least one MYC protein selected from c-MYC,c-MYC(T58A) and L-MYC that is repeatedly transfected into a cellcomprising said cell, tissue or organism once per day (at a total dailydose of about 0.6-1.2 μg per approximately 10⁵ cells (together with saidAgent mRNA) for approximately 15-20 days, said cell is reprogrammed froma somatic cell (e.g., a fibroblast or keratinocyte) to an iPS cell. Insome other preferred embodiments, wherein said Exogenous mRNA encodesMYOD1 that is repeatedly transfected into a cell comprising said cell,tissue or organism once per day for at least two days (e.g., at a totaldaily dose and in conjunction with the Agent mRNA as shown in theExamples herein), said cell is reprogrammed (e.g., differentiated ortransdifferentiated) from a mesenchymal stem cell or a somatic cell to amyoblast cell. In still other embodiments, said transfection of saidcell with said Exogenous mRNA (in conjunction with the Agent mRNA)results in transdifferentiation of one cell type into a second celltype, or differentiation of an iPSC, embryonic stem cell, orlineage-restricted stem cell into one or more specific downstreamlineages.

In certain embodiments, the transfecting of Exogenous RNA is conductedwithin 24 hours of the introducing of the Agent mRNA (e.g., from 1-24hours after the introducing or from 2-15 hours from the introducing). Incertain embodiments, the agent is mRNA in introduced at a level between0.1 and 3.5 μg/ml (e.g., 0.1 . . . 0.9 . . . 1.3 . . . 1.7 . . . 2.3 . .. 2.7 . . . 3.0 . . . 3.3 . . . or 3.5 μg/ml). In additionalembodiments, the Agent mRNA is introduced to the cells at a levelbetween 0.1 and 0.8 μg/ml. In particular embodiments, the cell ispresent in a medium, and the Agent mRNA results in synthesis of proteinthat is present in said medium. In further embodiments, the Agent mRNAresults in synthesis of protein that is present in the medium at a levelbetween 50 and 400 ng/ml (e.g., 50 . . . 100 . . . 150 . . . 200 . . .250 . . . 300 . . . 350 . . . or 400 ng/ml). In further embodiments, theAgent mRNA results in synthesis of protein that is present in saidmedium at a level between 100 and 300 ng/ml or about 200 ng/ml (e.g.,100 . . . 130 . . . 170 . . . 200 . . . 245 . . . 275 . . . or 300ng/ml).

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded as examples to further demonstrate certain aspects of thepresent invention. The invention may be better understood by referenceto one or more of these figures in combination with the detaileddescription of specific embodiments presented herein, but are notintended to limit the invention.

FIG. 1 shows that purified recombinant B18R protein added to thetransfection medium increases expression of transfected Exogenousfirefly luciferase mRNA. Firefly luciferase mRNA was transfected into BJfibroblast cells in medium containing various concentrations of purifiedrecombinant B18R protein. Cells were lysed and luciferase activity inlight units per microgram (LU/μg) of total protein was measured 20 hoursafter the luciferase mRNA transfection.

FIG. 2 shows that B18R protein is a secreted factor that increasesexpression of transfected Exogenous firefly luciferase (luc2) mRNA. A)Medium was conditioned for 48 hours after transfecting 1079 fibroblastcells with a DNA plasmid that expresses B18R protein. Collectedconditioned medium (CM) or medium from 1079 fibroblast cells that werenot transfected with the DNA plasmid that expresses B18R protein wasadded to fresh plates of 1079 fibroblast cells, which were thentransfected with Exogenous firefly luciferase mRNA. Cells were lysed andluciferase assays were performed 24 hours after the luciferase mRNAtransfections. Luciferase activity (LU/μg total protein) is shown for1079 fibroblast cells that were transfected with the Exogenousluciferase mRNA and then cultured in either B18R-conditioned medium ornon-B18R-conditioned medium. Mock-transfected cells were treated withthe transfection reagent but without Exogenous luciferase mRNA. B)Similar experiments to those described in A) were performed with a BJfibroblast cell line. Luciferase activity (LU/μg total protein) wasassayed 24 hours after transfecting the BJ fibroblast cells withExogenous firefly luciferase mRNA in B18R-conditioned medium ornon-B18R-conditioned medium.

FIG. 3 shows that introduction of an Agent mRNA comprising B18R mRNAboosts expression of Exogenous firefly luciferase (luc2) mRNA. A) 1079fibroblasts were first transfected with the indicated amount of an AgentmRNA comprising B18R mRNA. Then, after 18 to 20 hours, Exogenous fireflyluciferase mRNA was co-transfected along with an additional 1 μg of theAgent mRNA comprising B18R mRNA. Cells were lysed and luciferase assayswere performed 20 hours after luciferase mRNA transfections. Mocktransfected cells were only treated with the transfection reagentwithout Exogenous mRNA. B) Similar experiments were performed with a BJfibroblast cell line, except that the additional Agent mRNA comprisingB18R mRNA was omitted from the transfections with Exogenous mRNAcomprising luciferase mRNA.

FIG. 4 shows that the optimal length of time for introducing an AgentmRNA comprising B18R mRNA prior to transfection with an Exogenous mRNAcomprising firefly luciferase mRNA in order to obtain maximal expressionof said Exogenous mRNA is 24 hours or less. 1079 fibroblast cells weregrown for various times after introducing to the cells an Agent mRNAcomprising B18R mRNA. At various time points after said introducing ofthe Agent mRNA, the cells were transfected with Exogenous fireflyluciferase mRNA or mock transfected with only the transfection reagent,and then the cells were lysed 24 hours later and assayed for luciferaseactivity (LU/μg total protein). The increase in the expression of theExogenous mRNA was greatest if the Agent mRNA comprising B18R mRNA wasintroduced to the cells 24 or less hours prior to the transfection withExogenous firefly luciferase mRNA. Introducing the B18R mRNA to thecells at a time longer than 24 hours prior to transfecting the cellswith the firefly luciferase mRNA did not increase the amount of boost toluciferase activity conferred by the B18R mRNA.

FIG. 5 shows that introducing an Agent mRNA comprising B18R mRNAinhibits type I but not type II interferon activity. An innate immuneresponse reporter Hela cell line with stably integrated InterferonStimulated Response Elements (ISRE) upstream of firefly luciferase wastransfected with an Agent mRNA comprising B18R mRNA (0.5 μg/ml) or anegative control comprising EGFP mRNA (0.5 μg/ml), followed by treatment8 hours later with recombinant INFα (2777 U/ml), INFβ (333 U/ml) or INFγ(300 ng/ml) proteins. The luciferase assays were performed 16 hoursafter the addition of recombinant interferons to the cell culture media.In this example, luciferase activity indicates induction of an innateimmune response.

FIG. 6 shows that co-transfection of Agent mRNA comprising either E3L orK3L mRNA (which encode vaccinia virus E3L and K3L protein inhibitors ofthe interferon innate immune response pathway), enhance activity of anExogenous mRNA comprising Alkaline Phosphatase (ALKP) reporter mRNA. InFIG. 6.A), 0.2 μg/ml of Exogenous mRNA comprising ALKP mRNA wastransfected into mouse C3H10T1/2 mesenchymal stem cells, either alone ortogether with an Agent mRNA comprising 0.5 μg/ml of E3L mRNA, K3L mRNA,or both E3L and K3L mRNAs (each at 0.5 μg/ml), or with 0.5 μg/ml of EGFPmRNA as a negative control for the Agent mRNA comprising E3L or K3LmRNA. In FIG. 6.B), 0.2 μg/ml of Exogenous mRNA comprising ALKP mRNA wastransfected into human 1079 foreskin fibroblasts, either alone ortogether with an Agent mRNA comprising 0.5 μg/ml of E3L mRNA, K3L mRNA,or both E3L and K3L mRNAs (each at 0.5 μg/ml), or with 0.5 μg/ml of EGFPas a negative control for the Agent mRNA comprising E3L or K3L mRNA.Cells were lysed and ALKP reporter assays were performed 18 hours posttransfection (A, B).

FIG. 7 shows that an Agent mRNA comprising E3L mRNA inhibitsdsRNA-induced interferon activity in Hela cells. The ISRE Hela cell linewas transfected with 0.2 μg/ml of LIN28 dsRNA alone or together witheither 0.5 μg/ml of the Agent mRNA comprising E3L mRNA, or 0.5 μg/ml ofcMYC mRNA as a negative control for the Agent mRNA comprising E3L mRNA.Luciferase activity assays (LU/μg total protein) were performed 18 hourspost transfection.

FIG. 8 shows that myoblasts were induced from C3H10T1/2 mesenchymal stemcells that were co-transfected once per day for two days with ExogenousRNA comprising MYOD mRNA (0.6 μg/ml) and an Agent mRNA comprising E3LmRNA (5 μg/ml), K3L mRNA (5 μg/ml), or both E3L mRNA and K3L mRNA (0.5μg/ml of each), as shown by the Red immunofluorescence staining forMyosin Heavy Chain (MHC), a marker of muscle differentiation, in PanelsE, F and G. FIG. 8.A) shows untreated C3H10T1/2 cells. FIG. 8.B) showsmock-transfected cells. FIG. 8.C) shows cells transfected with only MYODmRNA. FIG. 8.D) shows cells co-transfected twice with MYODmRNAcells+EGFP mRNA (0.5 μg/ml) as a negative control in place of anAgent mRNA. FIG. 8.E) shows cells co-transfected twice with MYOD mRNA+anAgent mRNA comprising E3L mRNA. FIG. 8.F) shows cells co-transfectedtwice with MYOD mRNA+an Agent mRNA comprising K3L mRNA. FIG. 8.G) showscells co-transfected twice with MYOD mRNA+E3L and K3L mRNA.

FIG. 9 shows: (A) the nucleic acid sequence of B18R mRNA (SEQ ID NO:2)and (B) the amino acid sequence of B18R protein (SEQ ID NO:3).

FIG. 10 shows: (A) the nucleic acid sequence of E3L mRNA (SEQ ID NO:4)and (B) the amino acid sequence of E3L protein (SEQ ID NO:5).

FIG. 11 shows: (A) the nucleic acid sequence of K3L mRNA (SEQ ID NO:6)and (B) the amino acid sequence of K3L protein (SEQ ID NO:7).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods, kits, and compositions forincreasing translation of Exogenous mRNA in cells using an Agent mRNAthat reduces or suppresses an innate immune response induced, forexample, by the introduction of Exogenous mRNA. In certain embodiments,the Agent mRNA encodes a protein that inhibits or reduces type Iinterferon-induced cellular toxicity and/or inhibition of translationresulting from the introduction of Exogenous mRNA into a human or animalcell.

Definitions and General Aspects of the Invention

If the same terms or similar terms have been used with different meaningby others, including those cited in the section entitled “Background”herein, the terms when used to describe the present invention, shallnevertheless be interpreted to have the meanings presented below and inthe sections related to the specification and claims, unless otherwiseexpressly stated to the contrary.

When used in describing an aspect of the present invention, the terms“such as,” “including,” “for example,” “e.g.,” and the like shall beinterpreted to mean “without limitation.”

As used herein, an “Agent mRNA” means in vitro-synthesized mRNA encodingone or more proteins that affect the induction, activity or response ofan innate immune response pathway, whereby the innate immune response ina cell (e.g., a cell in a tissue or organism) is reduced, suppressed orprevented compared to the innate immune response in the absence ofintroducing said in vitro-synthesized mRNA.

As used herein, “Exogenous RNA” means RNA that is synthesized in an invitro transcription reaction by an RNA polymerase using a DNA templatethat exhibits an RNA polymerase promoter sequence recognized by said RNApolymerase upstream of a DNA sequence encoding the sequence of an RNAwhich is desired to cause a biological or medical effect, which effectdoes not include functioning as an Agent mRNA, and wherein said RNAinduces an innate immune response upon introduction into a cell, tissueor organism; “Exogenous RNA” includes, for example, RNA that exhibits asequence encoding at least one protein and which is capable of beingtranslated into protein upon introduction into a living cell that has afunctional translation system, and also includes RNA that exhibits anmRNA cap structure and a poly(A) tail. “Exogenous RNA” may alsoinclude-undesired RNA molecules that are synthesized in the in vitrotranscription reaction, including truncated RNA due to abortivetranscription or incomplete synthesis, uncapped in vitro transcriptionproducts, and dsRNA. In some cases herein, we refer to Exogenous RNAwhich encodes at least one protein (e.g., one or more proteins),including wherein the Exogenous RNA exhibits a cap structure and apoly(A) tail, as “Exogenous mRNA.” One important benefit of the methods,kits and compositions of the present invention is that the Agent mRNAreduces or suppresses an innate immune response which would be inducedby the Exogenous RNA in the absence of such Agent mRNA.

As used herein, “Exogenous siRNA” and “Exogenous miRNA” mean a siRNA ormiRNA, respectively, that is synthesized in vitro using any method knownin the art, and that is for the purpose of causing a biological ormedical effect in a cell, tissue or organism into which it isintroduced, which effect does not include functioning as an Agent mRNA.In some embodiments, the Exogenous miRNA or Exogenous siRNA issynthesized by in vitro transcription of a DNA template, including ineither one or two in vitro transcription reactions using either one ortwo DNA templates or one RNA polymerase that recognizes one RNApolymerase promoter sequence or two different RNA polymerases, each orwhich recognizes a different RNA polymerase promoter sequence, or bychemical synthesis on an oligonucleotide synthesizer using methods knownin the art.

Agent mRNA and Exogenous RNA (e.g., mRNA) can be made using similarmethods. For example, in some embodiments of the methods, kits, systemsor compositions of the invention, the Agent mRNA or Exogenous RNA, orother RNA, is synthesized by in vitro transcription (IVT) of a DNAtemplate using an RNA polymerase (e.g., SP6, T3 or T7 RNA polymerase)and nucleoside-5′-triphosphates (NTPs). In some embodiments, the NTPsused for IVT comprise or consist of only GTP ATP, UTP, and CTP(“canonical NTPs”), and the Agent mRNA or Exogenous RNA product isdescribed as “GAUC.” In other embodiments, a modified NTP is used inplace of some or all of one or more of the respective canonical NTPs. Insome preferred embodiments, the modified NTP,pseudouridine-5′-triphosphate (ψTP) is used for IVT in place of some orall of the UTP; if ψTP is used for IVT in place of all of the UTP, theAgent mRNA or Exogenous RNA product is described as “GAΨC.” In somepreferred embodiments, the modified NTP,5-methylcytidine-5′-triphosphate (m⁵CTP or 5mCTP) is used for IVT inplace of some or all of the CTP. In some preferred embodiments whereinψTP is used for IVT in place of some or all of the UTP, m⁵CTP is alsoused in place of some or all of the CTP. In some preferred embodiments,both ψTP and m⁵CTP are used for IVT in place of all of the correspondingUTP and CTP, and the Exogenous RNA product is described as “GAψm⁵C” or“GAψ5mC.” In most embodiments, the Agent mRNA or Exogenous RNA is mRNA,meaning that it exhibits a “cap” on its 5′-terminus and a poly(A) tailon its 3′-terminus, as will be generally understood by those withknowledge in the art.

In some preferred embodiments, Agent mRNA or Exogenous RNA that is mRNAis synthesized by IVT, followed by addition of the cap using a cappingenzyme system comprising RNA guanyltransferase activity and addition ofa poly(A) tail using a poly(A) polymerase (e.g., using an T7 mScript™Standard mRNA Production System, as described elsewhere herein). In someother embodiments, the cap is added by incorporation of a dinucleotidecap analog (e.g., m7GpppG or the 3′-O-methyl-m7GpppG ARCA) during IVT.In some embodiments, the poly(A) tail is added to the 3′-terminus duringIVT of a DNA template that encodes the poly(A) tail.

In some preferred embodiments of the methods, kits, systems andcompositions, the Agent mRNA, Exogenous RNA (e.g., Exogenous mRNA),Exogenous miRNA or Exogenous siRNA comprises or consists of GAψC RNA. Inother preferred embodiments of the methods, kits, systems andcompositions, the Agent mRNA, Exogenous RNA (e.g., Exogenous mRNA),Exogenous miRNA or Exogenous siRNA comprises or consists of GAψm⁵C RNA.

In some preferred embodiments, Agent mRNA is further purified. In someembodiments, Exogenous RNA (e.g., Exogenous mRNA) is also furtherpurified, in which embodiments, the same purification methods, purityquality standards, and assays for purity, as described herein may beused. In certain embodiments, the Agent mRNA is purified so that themRNA is substantially free, virtually free, essentially free, or free ofcontaminants (or of a particular RNA contaminant, such as dsRNA). By“substantially free,” “virtually free,” “essentially free,” or “free” ofcontaminants (or of a particular RNA contaminant, such as dsRNA), it ismeant that less than 0.5%, less than 0.1%, less than 0.05%, or less than0.01%, respectively, of the total mass or weight of the RNA in the AgentmRNA is composed of contaminants (or of a particular RNA contaminant,such as dsRNA). The amounts and relative amounts of non-contaminant mRNAmolecules and RNA contaminant molecules (or of a particular RNAcontaminant, such as dsRNA) may be determined by HPLC or other methodsused in the art to separate and quantify RNA molecules. In somepreferred embodiments wherein the Agent mRNA (including GAUC, GAψC orGAψm⁵C Agent mRNA) is substantially free, virtually free, essentiallyfree, or free of contaminant dsRNA, the relative amounts ofnon-contaminant mRNA and of contaminant dsRNA are assayed using the J2dsRNA-specific antibody (English & Scientific Consulting, Szirák,Hungary); by “substantially free,” “virtually free,” “essentially free,”or “free” of dsRNA it is meant that less than 0.5%, less than 0.1%, lessthan 0.05%, or less than 0.01%, respectively, of the total mass orweight of the RNA in the Agent mRNA consists of dsRNA of a size greaterthan about 40-basepairs in length when assayed by dot blot immunoassayas described below using the J2 dsRNA-specific antibody or using anotherassay that gives equivalent results to the assay described herein. Itshall be understood herein that the results of the dot blot immunoassaysusing the J2 dsRNA-specific antibody will be based on comparing theassay results obtained using the Agent mRNA with the assay results of J2dsRNA-specific antibody dot blot immunoassays performed at the same timewith dsRNA standards comprising known quantities of dsRNA of the same orequivalent size and J2 antibody binding.

As defined herein, Agent mRNA (or Exogenous mRNA) may be analyzed forthe amount or relative amount of contaminant dsRNA by performing thefollowing dot blot immunoassay using a dsRNA-specific antibody, such asthe J2 dsRNA-specific antibody, or another antibody that givesequivalent results: RNA samples are spotted (5μ1/dot) on NYTRAN SPCpositively charged nylon membranes and then allowed to dry on the nylonmembrane for 30 minutes. The membrane is then blocked in blocking buffer(25 mM Tris, pH 7.5, 150 mM NaCl, 0.05% TWEEN 20, 5% W/V dry milk) atroom temperature for 1 hour on a rotating platform. The primary antibody(e.g., J2 antibody; English & Scientific Consulting, Hungary) is addedat 1 μg/ml in blocking buffer at room temperature for 1 hour on arotating platform. The membranes are then washed 6 times for 5 minutesin 20 mls of wash buffer (25mM Tris, pH 7.5, 150 mM NaCl, 0.05% TWEEN20). The secondary antibody (anti-mouse HRP (Cell SignalingTechnologies, Danvers, Mass.) is added at 1:1000 in blocking buffer atroom temperature for 1 hour on a rotating platform. The membranes arethen washed 6 times for 5 minutes in 20 mls of wash buffer (25 mM Tris,pH 7.5, 150 mM NaCl, 0.05% TWEEN 20). Then, equal volumes of SUPERSIGNALWest Pico Chemiluminescent Substrates (Cat # 34080, Thermo Scientific)are added and the color is allowed to develop for 5 minutes on arotating platform. The dots are imaged by exposing film in the dark roomand developing the film in Kodak Developer for 1 minute and Kodak Fixerfor 1 minute.

The present invention is not limited with respect to the purificationmethods used to purify the Agent mRNA or Exogenous mRNA, and theinvention includes use of any method that is known in the art ordeveloped in the future in order to purify the mRNA and removecontaminants, including RNA contaminants, that interfere with theintended use of the mRNA. For example, in preferred embodiments, thepurification of the mRNA removes contaminants that are toxic to thecells (e.g., by inducing an innate immune response in the cells, or, inthe case of RNA contaminants comprising dsRNA, by inducing an interferonresponse or by inducing RNA interference (RNAi), e.g., (via siRNA, miRNAor long RNAi molecules) and contaminants that directly or indirectlydecrease translation of the mRNA in the cells. In some embodiments, themRNA is purified by HPLC. In certain embodiments, the mRNA is purifiedusing on a polymeric resin substrate comprising a C18 derivatizedstyrene-divinylbenzene copolymer and a triethylamine acetate (TEAA) ionpairing agent is used in the column buffer along with the use of anacetonitrile gradient to elute the mRNA and separate it from the RNAcontaminants in a size-dependent manner; in some embodiments, the mRNApurification is performed using HPLC, but in some other embodiments agravity flow column is used for the purification. In some embodiments,the mRNA is purified using a method described in the book entitled “RNAPurification and Analysis” (Gjerde et al., 2009). In some embodiments,the mRNA purification is carried out in a non-denaturing mode (e.g., ata temperature less than about 50° C., e.g., at ambient temperature). Insome embodiments, the mRNA purification is carried out in a partiallydenaturing mode (e.g., at a temperature less than about 50° C. and 72°C.). In some embodiments, the mRNA purification is carried out in adenaturing mode (e.g., at a temperature greater than about 72° C.).Those with knowledge in the art will know that the denaturingtemperature depends on the melting temperature (Tm) of the mRNA that isbeing purified as well as on the melting temperatures of RNA, DNA, orRNA/DNA hybrids which contaminate the mRNA. In some other embodiments,the mRNA is purified as described (Mellits et al., 1990). These authorsused a three step purification to remove the contaminants which may beused in embodiments of the present invention. Step 1 was 8%polyacrylamide gel electrophoresis in 7M urea (denaturing conditions).The major RNA band was excised from the gel slice and subjected to 8%polyacrylamide gel electrophoresis under nondenaturing condition (nourea) and the major band recovered from the gel slice. Furtherpurification was done on a cellulose CF-11 column using an ethanol-saltbuffer mobile phase which separates double stranded RNA from singlestranded RNA (Barber, 1966; Franklin, 1966; Zelcer et al., 1981) and thefinal purification step was cellulose chromatography. In some otherembodiments, the mRNA is purified using an hydroxylapatite (HAP) columnunder either non-denaturing conditions or at higher temperatures asdescribed in (Andrews-Pfannkoch et al., 2010; Clawson and Smuckler,1982; Lewandowski et al., 1971; Pays, 1977). In some other embodiments,the mRNA is purified by weak anion exchange liquid chromatography undernon-denaturing conditions as described by (Easton et al., 2010). In someembodiments, the mRNA is purified using a combination of any of theabove methods or another method known in the art or developed in thefuture. In still another embodiment, the mRNA used in the compositionsand methods of the present invention is purified using a process whichcomprises treating the mRNA with an enzyme that specifically acts on(e.g., digests) one or more contaminant RNA or contaminant nucleic acids(e.g., including DNA), but which does not act on (e.g., does not digest)the desired mRNA. For example, in some embodiments, the mRNA used in thecompositions and methods of the present invention is purified using aprocess which comprises treating the mRNA with a ribonuclease III (RNaseIII) enzyme (e.g., E. coli RNase III) and the mRNA is then purified awayfrom the RNase III digestion products. A ribonuclease III (RNase III)enzyme herein means an enzyme that digests dsRNA greater than abouttwelve basepairs to short dsRNA fragments. In some embodiments, the mRNAused in the compositions, kits and methods of the present invention ispurified using a process which comprises treating the mRNA with one ormore other enzymes that specifically digest one or more contaminant RNAs(e.g., dsRNA) or contaminant nucleic acids (e.g., including DNA).

“Differentiation” or “cellular differentiation” means the naturallyoccurring biological process by which a cell that exhibits a lessspecialized state of differentiation or cell type (e.g., a fertilizedegg cell, a cell in an embryo, or a cell in a eukaryotic organism)becomes a cell that exhibits a more specialized state of differentiationor cell type. Scientists, including biologists, cell biologists,immunologists, and embryologists, use a variety of methods and criteriato define, describe, or categorize different cells according to their“cell type,” “differentiated state,” or “state of differentiation.” Ingeneral, a cell is defined, described, or categorized with respect toits “cell type,” “differentiated state,” or “state of differentiation”based on one or more phenotypes exhibited by that cell, which phenotypescan include shape, a biochemical or metabolic activity or function, thepresence of certain biomolecules in the cell (e.g., based on stains thatreact with specific biomolecules), or on or in the cell (e.g., based onbinding of one or more antibodies that react with specific biomoleculesinside the cell or on the cell surface). For example, in someembodiments, different cell types are identified and sorted using a cellsorter or fluorescent-activated cell sorter (FACS) instrument.“Differentiation” or “cellular differentiation” can also occur to cellsin culture. In some embodiments, the term “reprogramming” is used hereinto refer to differentiation or cellular differentiation, includingde-differentiation or transdifferentiation, that occurs in response todelivery of one or more reprogramming factors into the cell, directly(e.g., by delivery of protein or polypeptide reprogramming factors intothe cell) or indirectly (e.g., by delivery of an exogenous RNApreparation of the present invention which consists of one or more mRNAmolecules, each of which encodes a reprogramming factor) and maintainingthe cells under conditions (e.g., medium, temperature, oxygen and CO₂levels, matrix, and other environmental conditions) that are conducivefor differentiation. The term “reprogramming” when used herein is notintended to mean or refer to a specific direction or path ofdifferentiation (e.g., from a less specialized cell type to a morespecialized cell type) and does not exclude processes that proceed in adirection or path of differentiation than what is normally observed innature. Thus, in different embodiments of the present invention,“reprogramming” means and includes any and all of the following:

-   -   (1) “Dedifferentiation”, meaning a process of a cell that        exhibits a more specialized state of differentiation or cell        type (e.g., a mammalian fibroblast, a keratinocyte, a muscle        cell, or a neural cell) going to a cell that exhibits a less        specialized state of differentiation or cell type (e.g., an iPS        cell);    -   (2) “Transdifferentiation”, meaning a process of a cell that        exhibits one specialized state of differentiation or cell type        (e.g., a mammalian fibroblast, a keratinocyte, or a neural cell)        going to a different specialized state of differentiation or        cell type (e.g., from a fibroblast or keratinocyte to a muscle        cell); and    -   (3) “Expected or Natural Differentiation”, meaning a process of        a cell that exhibits any particular state of differentiation or        cell type going to another state of differentiation or cell type        as would be expected in nature if the cell was present in its        natural place (e.g., in an embryo or an organism).

In some embodiments, the Agent mRNAs in the methods, compositions,systems, and kits of the present invention comprise or consist of theB18R, E3L, and K3L mRNAs that exhibit the nucleic acid sequences inFIGS. 9A-11A or that encode proteins that exhibit the amino acidsequences in FIGS. 9B-11B, as well as mRNAs that exhibit nucleic acidsequences or encode protein sequences that are variant sequences thatare substantially the same as those nucleic acid sequences or amino acidsequences. For example, one, two, or more bases in one, two, or morecodons may be changed in the nucleic acid sequence (or one, two or moreamino acids may be changed in the amino acid sequence) such that asequence differing from a sequence shown in any of FIGS. 9-11 isgenerated. Changes to the amino acid sequence may be generated bychanging the nucleic acid sequence encoding the amino acid sequence. Forexample, the mRNA encoding a variant of B18R, E3L, or K3L protein may beprepared by methods known in the art using the guidance of the presentspecification for particular sequences. These methods include, but arenot limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA template used for in vitrotranscription (IVT) of mRNA encoding a B18R, E3L, or K3L protein.Site-directed mutagenesis is a preferred method for preparingsubstitution variants. This technique is well known in the art (Carteret al., 1985; Kunkel, 1985).

Briefly, in carrying out site directed mutagenesis of a DNA template,the starting DNA is altered by first hybridizing an oligonucleotideencoding the desired mutation to a single strand of such starting DNA.After hybridization, a DNA polymerase is used to synthesize an entiresecond strand, using the hybridized oligonucleotide as a primer, andusing the single strand of the starting DNA as a template. Thus, theoligonucleotide encoding the desired mutation is incorporated into theresulting double-stranded DNA.

PCR mutagenesis is also suitable for making nucleic acid or amino acidsequence variants in the DNA template that is used for IVT (Vallette etal., 1989). Briefly, a small amount of the starting DNA template thatone wishes to mutate is amplified by PCR using at least one PCR primerthat exhibits a desired variant nucleic acid sequence compared to thecorresponding region in the starting DNA template to generate arelatively large quantity of a specific DNA fragment that differs fromthe starting DNA template sequence only at the positions where the atleast one PCR primers differed from the starting DNA template. This PCRmutagenesis process can be repeated using the product of a prior PCRmutagenesis reaction to introduce additional desired mutations in theDNA template.

Another method for preparing sequence variants, known as cassettemutagenesis, is based on the technique described by (Wells et al.,1985). The starting material is the plasmid (or other vector) comprisingthe starting DNA template to be mutated. The codon(s) in the startingDNA template to be mutated are first identified. There should be aunique restriction endonuclease site on each side of the identifiedmutation site(s). If no such restriction sites exist, they are generatedin the starting DNA template using the above-describedoligonucleotide-mediated mutagenesis method. The plasmid DNA is then cutwith the restriction enzyme(s) to linearize it at these sites. Twooligonucleotides that exhibit the sequences of each strand of the DNAbetween the restriction sites but containing the one or more desiredmutations are synthesized using standard procedures, and then hybridizedtogether using standard techniques to generate a double-stranded DNAreferred to as the cassette. This cassette is designed to have 5′ and 3′ends that are compatible with the ends of the linearized plasmid, suchthat it can be directly ligated into the plasmid DNA from which thecorresponding unmutated DNA was removed. This plasmid DNA now containsthe mutated DNA sequence and can be used to prepare the DNA template forin vitro transcription of mRNA that exhibits the desired variantsequence.

Alternatively, or additionally, the desired amino acid sequence encodingone or more polypeptide variants can be determined, and a nucleic acidsequence encoding such amino acid sequence variant(s) can be generatedsynthetically. Conservative modifications in the amino acid sequences ofthe proteins may also be made. Naturally occurring residues are dividedinto classes based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Conservative substitutions will entail exchanging a member of one ofthese classes for another member of the same class. The expectedactivity of the variant protein is confirmed following introduction ofthe Agent mRNA variant (e.g., from in vitro transcription of the variantDNA template) into the cell using methods disclosed herein.

Variant Agent mRNAs that encode variant B18R, E3L, or K3L proteins aregenerated (e.g., by truncation, deletion, or insertion into the DNAtemplate for IVT) and screened as described in the Examples herein todetermine if they function to reduce or suppress the innate immuneresponse induced by a Foreign Substance (e.g., by the introduction of anExogenous RNA into a cell). In this regard, any variant of an Agent mRNAthat is constructed can be screened to identify variants suitable foruse as a composition of the present invention or for use in a kit ormethod of the present invention.

Some embodiments of the invention comprise a kit or compositioncomprising or consisting of the mRNA encoding the antibody or artificialantibody. In some embodiments of any of the methods, kits andcompositions of the invention, the mRNA is Agent mRNA encoding anantibody or artificial antibody. In some embodiments of any of themethods, kits and compositions of the invention, the mRNA is ExogenousmRNA encoding an antibody or artificial antibody.

Still another embodiment of the invention is a composition comprising orconsisting of Exogenous mRNA encoding an antibody or artificial antibodyfor any desired function for which an antibody comprising protein isused in a cell, tissue or organism. For example, in some embodiments,the Exogenous mRNA encodes one or more antibodies or artificialantibodies that binds to a cell-specific or disease-specific orpathogen-specific protein that is expressed in a human or animal cell,tissue or organism. For example, in some embodiments, the Exogenous mRNAencodes one or more antibodies or artificial antibodies that binds to acancer-specific or tumor-specific protein. In some embodiments of themethod, Exogenous mRNA encoding one or more antibodies or artificialantibodies that is or are specific for a condition, disease or pathogeninfecting a human or animal patient is administered to the patient totreat the condition, disease or pathogen-induced state (e.g., byadministering the Exogenous mRNA to a cell, tissue or organism in thepatient, e.g., by transfection, electroporation, or by intravenous,interperitoneal, intradermal, subdermal, or internodal injection). Insome embodiments, the sequence of an mRNA encoding an antibody thatreduces an innate immune response is first made in a non-human speciesand then, using any of the methods known in the art, the Agent mRNA inmade by modifying the sequence so that the protein encoded by said AgentmRNA is similar to an antibody which would be produced naturally inhumans; the antibody encoded by said Agent mRNA is then said to be“humanized” because it is has been adapted to be suitable for use inhumans with minimal chance of inducing an active immune response. AgentmRNA encoding antibodies intended for use in other species can besimilarly adapted for use in those species.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

These examples demonstrate that Agent mRNA comprising or consisting ofB18R mRNA, E3L mRNA, and K3L mRNA, alone or in combination, decreasecell toxicity (or increase cell survival) and increase translation ofExogenous mRNAs that are transfected into human or other mammaliancells. For example, introducing these Agent mRNAs into cells at the sametime as or prior to (e.g., in some embodiments, 8-24 hours prior to)transfecting the cells with Exogenous mRNAs encoding one or more otherproteins of interest enhances the translation or activity of theproteins encoded by those other Exogenous mRNAs. These Examples alsodemonstrate that introducing these Agent mRNAs into cells inhibits thebiological activity of type I interferons (IFNα, IFNβ) but not type IIinterferons (INFγ). The Examples also demonstrate that introducing AgentmRNAs comprising E3L mRNA, K3L mRNA, or both E3L mRNA and K3L mRNAtogether with an Exogenous mRNA comprising in vitro-transcribed MYODmRNA resulted in reprogramming of mouse C3H10T1/2 mesenchymal stem cellsto myoblast cells, whereas no reprogramming occurred when the C3H10T1/2mesenchymal stem cells were transfected only with Exogenous mRNAcomprising MYOD mRNA in the absence of these Agent mRNAs.

Materials and Methods

Templates for In Vitro Transcription

A B18R DNA template for preparing Agent mRNA comprising or consisting ofB18R mRNA (GAψC) was prepared as follows: a B18R coding sequence (cds)was cloned into a pUC-based plasmid DNA that contained a T7 RNApolymerase promoter followed by 5′ Xenopus Beta Globin (UTR), a cloningsite (into which the B18R cds was inserted), and a 3′ Xenopus BetaGlobin 3′ UTR, and then linearized with NotI.

An E3L DNA template for preparing Agent mRNA comprising or consisting ofE3L mRNA (GAψm⁵C) was prepared as follows: an E3L cds was cloned into apUC19-based plasmid DNA that contained a T7 RNA polymerase promoterfollowed by 5′ Xenopus Beta Globin (UTR), a cloning site (into which theE3L cds was inserted), and a 3′ Xenopus Beta Globin 3′ UTR, and thenlinearized with SalI.

A K3L DNA template for preparing Agent mRNA comprising or consisting ofK3L mRNA (GAψm⁵C) was prepared as follows: a K3L cds was cloned into apUC19-based plasmid DNA that contained a T7 RNA polymerase promoterfollowed by 5′ Xenopus Beta Globin (UTR), a cloning site (into which theK3L cds was inserted), and a 3′ Xenopus Beta Globin 3′ UTR, and thenlinearized with SalI.

EGFP and c-MYC DNA templates for preparing EGFP or c-MYC mRNA (GAψm⁵C),respectively, for use as negative control mRNAs in place of Agent B18R,E3L or K3L mRNAs, were prepared as follows: the respective EGFP or c-MYCcds was cloned into a pUC19-based plasmid DNA that contained a T7 RNApolymerase promoter followed by 5′ Xenopus Beta Globin (UTR), a cloningsite (into which the respective cds was inserted), and a 3′ Xenopus BetaGlobin 3′ UTR, and then linearized with SalI.

A mouse Alkaline Phosphatase (ALKP) DNA template for preparing ExogenousmRNA comprising or consisting of mouse ALKP mRNA (GAUC) was prepared asfollows: the mouse ALKP cds was cloned into a pUC19-based plasmid DNAthat contained a T7 RNA polymerase promoter followed by 5′ Xenopus BetaGlobin (UTR),

a cloning site (into which the mouse ALKP cds was inserted), and a 3′Xenopus Beta Globin 3′ UTR, and then linearized with SalI.

A mouse MYOD DNA template for preparing mouse Exogenous mRNA comprisingor consisting of mouse MYOD mRNA (GAUC) for use in reprogramming mousemesenchymal stem cells or somatic cells (e.g., fibroblasts) to myoblastcells was prepared as follows: the MYOD cds was cloned into apUC19-based plasmid DNA that contained a T7 RNA polymerase promoterfollowed by 5′ Xenopus Beta Globin (UTR), a cloning site (into which theMYOD cds was inserted), and a 3′ Xenopus Beta Globin 3′ UTR, and thenlinearized with SalI.

A luciferase (luc2) DNA template for preparing Exogenous mRNA comprisingor consisting of firefly luciferase luc2 (Photinus pyralis luc2) mRNA(GAUC) was obtained by linearizing a commercially available plasmid(Promega, Madison, Wis.).

DNA templates for preparing other Exogenous mRNAs are similarly preparedas follows: the cds is cloned into a pUC19-based plasmid DNA thatcontains a T7 RNA polymerase promoter followed by 5′ Xenopus Beta Globin(UTR), a cloning site (into which the cds is inserted), and a 3′ XenopusBeta Globin 3′ UTR, and then linearized with SalI (or anotherrestriction enzyme if the cds contains a SalI restriction site). Forexample, DNA templates were thus prepared for use in making mRNAsencoding the human and mouse transcription factors OCT4, SOX2, KLF4,LIN28, NANOG, c-MYC, L-MYC, and c-MYC(T58A), and Exogenous mRNAs areprepared using these templates as described herein, and used forreprogramming somatic cells to pluripotent stem cells. For example, insome embodiments, a total of about 1 to 1.2 microgram per transfectionof mRNAs encoding human or mouse OCT4, SOX 2, KLF4 and a MYC proteinselected from c-MYC, L-MYC and c-MYC(T58A) at a ratio of 3:1:1-3:1,respectively, (or 1 to 1.2 microgram per transfection of mRNAs encodinghuman or mouse OCT4, SOX 2, KLF4 and a MYC protein selected from c-MYC,L-MYC and c-MYC(T58A), and LIN28 and/or NANOG at a ratio of3:1:1-3:1:1:(1) are transfected into human or animal somatic cells oncedaily for about 18 days, whereby the somatic cells are reprogrammed topluripotent stem cells. In some embodiments, the use of an Agent mRNAencoding B18R, E3L and/or K3L proteins, or encoding other proteinsinhibitors of innate immune response that are disclosed herein,facilitates or enhances the reprogramming of somatic cells topluripotent stem cells by these Exogenous mRNAs. In still otherembodiments, said Agent mRNA facilitates or enhances the reprogramming(e.g., differentiation, transdifferentiation) of one type of cell toanother type of cell).

In Vitro Transcription, Capping and Polyadenylation to Make mRNAs

Each of the mRNAs used for transfection in the various experiments wasprepared by in vitro transcription of the respective linear DNA templateusing the in vitro transcription components, the capping enzymecomponents, and the polyadenylation components provided in a T7 mScript™Standard mRNA Production System (or the IVT components of an INCOGNITO™T7 Ψ-RNA Transcription Kit or an INCOGNITO™ T7 5mC- & Ψ-RNATranscription Kit) as described by the manufacturer (CELLSCRIPT, Inc.,Madison, Wis.) unless otherwise stated herein. In some experiments inwhich a T7 mScript™ Standard mRNA Production System was used,pseudouridine triphosphate (ΨTP) and/or m⁵CTP was used for in vitrotranscription instead the corresponding UTP or CTP, respectively. TheDNA templates for in vitro transcription were prepared as generallydescribed in the T7 mScript™ or INCOGNITO™ product literature (e.g., bylinearization of plasmid containing the mRNA coding sequence or by PCRof said gene).

NotI-linearized B18R DNA template was used as a template in in vitrotranscription reactions using either the INCOGNITO™ T7 Ψ-RNATranscription Kit, which contains pseudouridine triphosphate (ΨTP)instead of UTP (CELLSCRIPT, Inc.), or the in vitro transcriptioncomponents in the T7 mScript™ Standard mRNA Production System, exceptthat ΨTP was used in place of the UTP, to generate GAΨC RNA, which wassubsequently capped and tailed to make GAΨC mRNA.

E3L or K3L DNA templates were used as templates in in vitrotranscription reactions containing ΨTP and m⁵CTP to generate GAΨm⁵CRNAs, which were subsequently capped and tailed to make GAΨm⁵C mRNAs.Similarly, in some experiments, EGFP RNA or c-MYC mRNA was made for useas a negative control in place of Agent B18R, E3L or K3L mRNA by invitro transcription of the respective linearized DNA template usingeither an INCOGNITO™ T7 5mC- & Ψ-RNA Transcription Kit (CELLSCRIPT,Inc.), which contains m⁵CTP and ΨTP, or the T7 mScript™ Standard mRNAProduction System, but with m⁵CTP (Trilink, San Diego, Calif.) and ΨTPin place of standard CTP and UTP, respectively; these were subsequentlycapped and tailed to make GAΨm⁵C mRNAs.

Prior to capping and poly(A) tailing, Agent mRNAs E3L mRNA and K3L mRNA,as well as EGFP and c-MYC mRNAs, which were used as negative controls inplace of Agent mRNA in the Examples herein, were treated with RNase IIIto remove interferon-inducing dsRNA, as described in the literatureprovided with the MINiMMUNE™ Kit (CELLSCRIPT, Inc).

All mRNAs used herein as Exogenous mRNAs that were not Agent mRNAs forinhibiting an innate immune response or control mRNAs for replacing anagent comprising mRNA for inhibiting an innate immune response were madeby in vitro transcription of the respective DNA templates using the IVTcomponents of the T7 mScript™ Standard mRNA Production System and onlythe canonical nucleotides GTP, ATP, UTP and CTP (GAUC). For example,firefly luciferase luc2 mRNA and mouse ALKP mRNA, which were used asExogenous mRNAs for expressing proteins in cells whose activities couldbe easily detected and quantified, and MYOD mRNA, which was used asExogenous mRNAs for expression in cells in order to induce reprogrammingof the cells to myoblasts, were made by in vitro transcription of therespective DNA templates using the IVT components of the T7 mScript™Standard mRNA Production System and only the canonical nucleotides GTP,ATP, UTP and CTP to generate GAUC RNA. For example, in some experiments,firefly luciferase luc2 (Photinus pyralis luc2) RNA was made for use asExogenous mRNA by in vitro transcription of a linearized plasmid(Promega, Madison, Wis.) with only GTP, ATP, UTP and CTP (i.e., withoutsubstitution by ΨTP or m⁵CTP). The Exogenous mRNAs containing GAUCdisclosed herein are solely for the purpose of examples and are notintended to limit the application of the methods, compositions or kitsdisclosed herein. For example, in other embodiments, these ExogenousmRNAs are made by in vitro transcription using ΨTP in place of UTP, andin still other embodiments, the Exogenous mRNAs are made by in vitrotranscription using m⁵CTP in place of CTP, including wherein ΨTP is usedin place of UTP.

In order to make mRNA, in vitro-transcribed RNAs are capped using theScriptCap™ m⁷G Capping Enzyme System (CELLSCRIPT, Inc.) to make cap0 RNAor using both the ScriptCap™ m⁷G Capping Enzyme System and theScriptCap™ 2′-O-Methyltransferase (CELLSCRIPT, Inc.) to make cap1 RNA,or with the same capping enzyme components in the T7 mScript™ StandardmRNA Production System; unless otherwise stated herein, all of thecapped mRNAs used in the Examples herein exhibited a cap1 structure. Forexample, B18R RNA was capped with the ScriptCap™ m⁷G Capping EnzymeSystem and ScriptCap™ 2′-O-Methyltransferase (CELLSCRIPT, Inc.) or withthe corresponding capping enzyme components in the T7 mScript™ StandardmRNA Production System, as described in the respective productliterature.

The resulting capped RNAs were polyadenylated using either the A-Plus™Poly(A) Polymerase Tailing Kit (CELLSCRIPT, Inc.) or the poly(A) tailingcomponents of the T7 mScript™ Standard mRNA Production System, asdescribed in the respective product literature. For example, theresulting Cap 1-capped B18R RNA was polyadenylated using either theA-Plus™ Poly(A) Polymerase Tailing Kit or the poly(A) tailing componentsof the T7 mScript™ Standard mRNA Production System to generate B18RmRNA. For example, a 30-minute reaction using the A-Plus™ Poly(A)Polymerase Tailing Kit generated mRNAs with a poly(A) tail comprisingapproximately 150 A residues.

The in vitro-transcribed and capped and poly(A)-tailed Agent mRNAs orExogenous mRNAs were made and purified as described in the literatureprovided with the T7 mScript™ Standard mRNA Production System. Briefly,after completion of the IVT reaction, the DNA template for IVT wasdigested by adding RNase-free DNase I to the in vitro transcriptionreaction and incubating for 15 minutes at 37° C.

Then, the RNA was phenol-chloroform extracted, then precipitated byadding an equal volume of 5M ammonium acetate, incubated on ice for 10minutes and spinning at 13,000 rpms for 10 minutes. The RNA pellet waswashed with 70% ethanol and dissolved in water. Following capping andpoly(A) tailing, the mRNA was again phenol-chloroform extracted,precipitated with ammonium acetate, washed with 70% ethanol anddissolved in water.

mRNA Transfections

Transfections were performed using commercially available transfectionreagents, including the TransIT™ mRNA transfection reagent (MirusBiosciences) and RNAiMax™ (Invitrogen), as described in themanufacturers' literature. For example,

for TransIT™, the RNA was diluted in 250 μls Opti-MEMI (Invitrogen) andmixed with 5 μls TransIT™ BOOST reagent and 5 μls TransIT™ transfectionreagent and the mixture was immediately applied to the cells. Thepresent invention is not limited to use of these transfection reagentsfor delivering the Agent mRNA into cells.Any reagent or method (e.g., electroporation) that results in efficientdelivery of the Agent mRNA into the cells and that does not result inhigh toxicity can be used in or with the compositions, kits or methodsof the present invention.

Luciferase Assays

In all experiments with firefly luciferase luc2, except thepre-treatment time course, cells were washed in 2 mls 1×PBS andincubated with 500 μls 1× Reporter Lysis Buffer (Promega). Plates werefrozen for at least 1 hour, as the buffer requires a freeze-thaw cycle,and thawed at room temperature. Lysates were then transferred intomicrocentrifuge tubes and activity was determined using the LuciferaseAssay System (Promega), where 20 μls of lysate was incubated with 100μls of the kit luciferase reagent. Light emission was measured for 10seconds with no lag time on a Lumiskan™ Ascent luminometer (ThermoScientific). Protein concentration was determined for lysate samplesusing the Pierce BCA Protein Assay Kit (Fisher) and used to determinethe total amount of protein present in 20 μls of lysate. Luminescencereadings were normalized to the amount of protein used for theluciferase assays. In the pre-treatment time course experiment, cellswere lysed using 1× Passive Lysis Buffer (Promega), in which a freezethaw cycle was not required. Cells in these experiments were washed with2 mls 1×PBS, incubated with 500 μls 1X Passive Lysis Buffer for 2minutes at room temperature, and transferred to microcentrifuge tubes.The lysate was then used in activity assays as described above.

Assays of the Effects of Purified B18R Protein on Expression ofExogenous

Luciferase mRNA

BJ fibroblasts (ATCC) were plated onto 6-well dishes coated with 0.1%gelatin (Millipore) at 1×10⁵ cells per well. Cells were fed fibroblastmedia consisting of Advanced MEM (Invitrogen), 10% Hyclone HeatInactivated FBS (Fisher), 2 mM GLUTAMAX™ (Invitrogen), and 0.1 mMbeta-mercaptoethanol (Sigma). Purified B18R protein (eBiosciences) wasadded to make final concentrations of 0, 50, 100, 200 or 400 ng/ml.Cells were transfected with luciferase mRNA at a final concentration of1.4 μg/ml as described above. After 20 hours, cells were lysed andassayed for luciferase activity as described above.

Assays for Effects of B18R-Conditioned Medium on Luciferase Expression

Either BJ or 1079 fibroblast cells (both from ATCC) were plated 1×10⁵cells per well in a 6-well dish coated with 0.1% gelatin. Both celltypes were transfected with a plasmid that expresses the B18R proteinunder control of the constitutive CMV promoter at a final concentrationof 2.7 μg/ml using Lipofectamine™ 2000 (Invitrogen). A control plasmidthat expressed EGFP under control of the CMV promoter was co-transfectedat 0.5 μg per reaction to check how well the transfection procedureworked. In the DNA transfections, 0.5 μl of Lipofectamine 2000 per μg ofDNA was mixed with 12.5 μls per μg DNA of Opti-MEMI and incubated atroom temperature for 5 minutes. The mixture was then added to a solutionof the DNA plus 12.5 μls per μgs of DNA of Opti-MEMI. The transfectionmix was incubated at room temperature for 20 minutes before applicationto cells fed with 1.5 mls of fibroblast media. Transfection medium wasremoved 4 to 5 hours after transfection, and cells were fed with 2.5 mlsfresh fibroblast media per well. In the case of 1079 cells, the mediumwas conditioned for 48 hours, while for the BJ fibroblasts, the mediumwas conditioned for 20 hours. Conditioned media were collected from thecells and fed to a new plate of the same cell type. Control conditionedmedium was made by transfecting cells with the same amount of theplasmid that expressed EGFP as was used to transfect the cells with theplasmid that expressed the B18R protein. Luciferase mRNA was transfectedinto the cells at a final concentration of 1.4 μg/ml in the presence ofthe conditioned medium, and cells were assayed for luciferase activity24 hours later according to the procedures described above. Mocktransfections with only the transfection reagent without any luciferasemRNA present were done as controls.

Assays for Effects of B18R mRNA on Luciferase Expression

Either BJ or 1079 fibroblast cells were plated at 1×10⁵ cells per wellin a 6-well dish coated with 0.1% gelatin. Both cell types weretransfected with various amounts of Agent mRNA consisting of B18R mRNAas indicated in FIG. 3 using the procedure described above. After 18 to20 hours, cells were transfected with luciferase mRNA at a finalconcentration of 1.4 μg/ml. For 1079 cells, 1 μg of B18R mRNA was alsoadded as a co-transfectant. However, since the additionalco-transfection of the 1079 cells with the Agent mRNA consisting of B18RmRNA did not increase luciferase expression compared to the B18R mRNApre-treatment only, the additional co-transfection was omitted with BJfibroblast cells and only the pre-treatment with the B18R mRNA was usedfor the experiment. Cells were lysed and assayed for luciferase activity20 hours after luciferase mRNA transfection using the proceduredescribed above.

Time Course of B18R Pre-treatment on Expression of Exogenous mRNA

1079 fibroblast cells were plated at 1 ×10⁵ cells per well in a 6-welldish coated with 0.1% gelatin. B18R mRNA was transfected at a finalconcentration of 0.4 μg /ml in fibroblast medium using the proceduredescribed above. At various time points, luciferase mRNA was transfectedat a final concentration of 1.4 μgs/ml as described above. Twenty-fourhours after luciferase mRNA transfection, cells were lysed as describedabove for luciferase assays and stored at −80 ° C. until all time pointswere collected. Luciferase activity was measured as described above.Mock transfections were done without any luciferase mRNA present in thetransfection mixes, and samples were collected after 24 and 48 hoursafter B18R mRNA transfection.

Use of a Hela Cell Line Containing ISREs to Assay B18R mRNA

Inhibition of Specific Interferon Responses

In some embodiments, Interferon Stimulated Response Elements (ISRE)(e.g., DNA containing four ISRE sites that exhibit the followingsequence (SEQ ID NO: 1):

5′-CAGTTTCACTTTCCCCAGTTTCACTTTCCCCAGTTTCACTTTCCCCA GTTTCACTT-3′are inserted into the XhoI and BglII sites of pGL4.26 plasmid (Promega,Madison, Wis.) upstream of a minimal promoter and the luc2 luciferasegene.

In some experiments, a human or animal cell line (e.g., a Hela humancell line) containing the ISREs is generated by using Lipofectamine™2000 (Invitrogen, Carlsbad, Calif.) to transfect the cells (e.g.,standard Hela cells from ATCC, Manassas, Va.) with thepGL4.264×ISREs-luc2-containing plasmid and clones in which theISREs-luc2 are integrated are isolated by serial dilution of cells in96-well dishes and selection with Hygromycin B (InivoGen, San Diego,Calif.; e.g., at 200 μg/ml), followed by confirmation that the celllines are responsive to recombinant Interferons (e.g., INFα cat#11100-1, INFβ cat#11415-1, and INFγ cat#285-IF from R&D Systems,Minneapolis, Minn.).

The ISREs-luc2 cell line is then used to assay the interferon responsethat results from the various interferons following transfection of thecell line with Agent B18R mRNA, compared to the interferon response thatresults following transfection of the cell line with EGFP mRNA as anegative control for the Agent mRNA comprising B18R mRNA. These resultsindicate if and to what extent the Agent B18R mRNA inhibits or reducesthe interferon response by each of the respective interferons (e.g.,INFα, INFβ or INFγ), thereby showing the specificity of interferonresponses and their levels of response at various times (e.g., 8 hours,16 hours, 24 hours) after transfection with Agent B18R mRNA compared tothe EGFP mRNA as a negative control for the Agent B18R mRNA (e.g., byperforming luciferase assays using the Bright-Glo™ Luciferase AssayReagent from Promega, Madison, Wis., and a SpectraMax M3 luminometerfrom Molecular Devices, Sunnyvale, Calif.). For example, in someexperiments, a Hela line containing the ISREs upstream of the luciferasegene are transfected with B18R mRNA or EGFP mRNA (e.g., each at 0.5μg-1/ml), followed by treatment 8 hours later with recombinant INFα□(2777 U/ml), INFβ□ (333 U/ml) or INFγ (300 ng/ml) proteins and assay forluciferase activity at various times (e.g., 8 hours, 16 hours, 24 hours,or 48 hours) after the addition of the recombinant interferons to thecell culture media.

Use of a Hela Cell Line Containing ISREs to Assay E3L mRNA

Inhibition of Specific Interferon Responses

The ISRE-luc2 Hela cell line was also used to test the effect of anAgent mRNA comprising E3L mRNA on the induction of an innate immuneresponse by co-transfected LIN28 dsRNA. The LIN28 coding sequence wascloned in a pUC29-based plasmid downstream of T7 and T3 RNA polymerasepromoters, and then different aliquots of the plasmid, which werelinearized with BamHI or EcoRI, respectively, were used as templates forin vitro transcription with T7 and T3 RNA polymerases, respectively, instandard in vitro transcription reactions using GAUC canonical NTPs.These complementary RNAs were hybridized to generate dsRNA using thefollowing hybridization parameters; 10 minutes at 70° C., 10 minutes at60° C., 10 minutes at 50° C., 10 minutes at 40° C., then allowing theRNA to anneal for another 30 minutes at room temperature (22° C.).RNAiMax™ (Invitrogen) was used to transfect ISRE-luc2 cells with 0.2μg/ml of LIN28 dsRNA along with 0.5 μg/ml of either Agent mRNAcomprising E3L mRNA or human c-MYC mRNA as a negative control for theAgent E3L mRNA. Bright-Glo™ luciferase assays were performed 18 hourspost transfection.

Effects of Agent mRNAs Comprising E3L or K3L mRNA on Expression ofExogenous mRNA Comprising Mouse Alkaline Phosphatase mRNA

In order to assay for the effects of Agent mRNA on expression ofExogenous mRNA consisting of mouse ALKP mRNA, 0.2 μg/ml of ALKP GAUCmRNA was transfected using RNAiMax into mouse C3H10T1/2 mesenchymal stemcells or 1079 human fibroblasts, either alone or together with 0.5 μg/mlof Agent mRNA comprising EGFP mRNA, E3L mRNA, K3L mRNA, or both E3L mRNAand K3L mRNA (each at 0.5 μg/ml). Cells were lysed and ALKP reporterassays were performed 18 hours post transfection. Absorbance was read ona spectrophotometer at 405 nm as a readout of ALKP activity.

Use of Agents Comprising E3L or K3L mRNA to Facilitate MYOD mRNA-InducedReprogramming of Mesenchymal Stem Cells to Myoblast Cells

Mouse C3H10T1/2 cells (Passage 16) were plated at 2×10⁵ cells per wellof a gelatin-coated 6-well dish and grown overnight in DMEM, 10% FBS,GLUTAMAX, and pen/strep. The next day, the cells were switched todifferentiation media (DMEM+2% horse serum, GLUTAMAX, and pen/strep).MYOD mRNAs were in vitro transcribed using the T7 mScript™ Standard mRNAProduction System with GAUC nucleotides while E3L, K3L, EGFP mRNAs wereall made using the T7 mScript™ RNA Transcription Kit with GAψm⁵Cnucleotides. Cells were transfected with 0.6 μg/ml of the MYOD GAUCmRNA, and an Agent mRNA comprising E3L GAΨm⁵C mRNA (5 μg/ml), K3L GAΨm⁵CmRNA (5 μg/ml), or both E3L GAΨm⁵C mRNA and K3L GAΨm⁵C mRNA (5 μg/ml ofeach) using RNAiMax in differentiation media. mRNA was added to a tubecontaining OptiMEM media with the total volume equaling 60 μl in tube A.5 μl of RNAiMax was added to tube B for every g mRNA totaling 60 μl intube B. Tube A and Tube B were mixed and incubated at room temperaturefor 15 minutes. After 15 minutes the mRNA/RNAiMax mix was added to 2 mlof differentiation media already on the cells. The media were changed 4hours post transfection with new differentiation media. Twenty-fourhours after the first transfection another MYOD mRNA transfection wasadministered. The media were again changed 4 hours post transfection.Forty-eight hours after the first transfection, the cells were fixed andimmunofluorescence assays were performed to detect Myosin Heavy Chain(MHC) expression, which is a marker for myoblast muscle differentiation.

Immunofluorescence.

C3H10T1/2 cell plates were washed with PBS and fixed in 4%paraformaldehyde in PBS for 30 minutes at room temperature. The cellswere then washed 3 times for 5 minutes each wash with PBS followed bythree washes in PBS+0.1% Triton X-100. The cells were then blocked inblocking buffer (PBS+0.1% Triton, 2% FBS, and 1% BSA) for 1 hour at roomtemperature. The cells were then incubated for 2 hours at roomtemperature with the primary antibody (mouse anti-human MHC Cat#05-716,Millipore, Temecula, Calif.), at a 1:1000 dilution in blocking buffer.After washing 5 times in PBS+0.1% Triton X-100, the C3H10T1/2 cells wereincubated for 2 hours with the anti-mouse Alexa Fluor 555 (Cat#4409,Cell Signaling Technology, Danvers, Mass.) at 1:1000 dilutions inblocking buffer. Images were taken on a Nikon TS 100F invertedmicroscope (Nikon, Tokyo, Japan) with a 2-megapixel monochrome digitalcamera (Nikon) using NIS-elements software (Nikon).

Results

B18R Protein Increased Activity of Firefly Luciferase mRNA in Cells.

BJ fibroblast cells transfected with luciferase mRNA in the presence ofvarying concentrations of purified recombinant B18R protein showed anincrease in luciferase activity compared to cells transfected in theabsence of B18R protein in the same medium (FIG. 1). Mock transfectedcells, meaning cells treated with transfection reagent alone, showed noluciferase activity. Luciferase activity increased as the concentrationof B18R protein increased up to 200 ng/ml. A drop in luciferase activitywas seen at a higher B18R concentration of 400 ng/ml. However the levelof luciferase activity at 400 ng/ml of B18R protein was still higherthan that in cells transfected in the absence of B18R protein.

Luciferase Activity by Luciferase mRNA is Increased in Cells ExpressingB18R Protein.

Two cells lines, BJ fibroblasts and 1079 fibroblasts, were tested forthe effects of transfecting luciferase mRNA using media conditioned bycells expressing B18R protein. In both BJ fibroblast (FIG. 2A) and 1079fibroblast (FIG. 2B) cell lines, luciferase activity was increased whenluciferase mRNA was transfected in the presence of media conditioned byB18R protein-expressing cells compared to cells transfected in mediaconditioned by EGFP-expressing cells as a negative control.Mock-transfected control cells, which were treated with transfectionreagent alone, showed no luciferase activity in either medium.

Introducing B18R mRNA Prior to Transfection of Luciferase mRNA IncreasedLuciferase Activity.

Transfection of B18R mRNA before transfection of luciferase mRNAincreased luciferase activity in both BJ fibroblast (FIG. 3A) and 1079fibroblast (FIG. 3B) cell lines. In 1079 cells, all doses of B18R mRNAtransfected 18 to 20 hours prior to luciferase mRNA transfectionresulted in an increase in luciferase activity, with the maximal boostseen at the 0.4 μg/ml dose of B18R mRNA (FIG. 3A). BJ fibroblasts alsoshowed an increase in luciferase activity when B18R was transfected intothe cells 18 to 20 hours before luciferase transfection (FIG. 3B). Atthe 1.4 μs/ml and 2.9 μs/ml doses of B18R mRNA, the BJ fibroblasts didnot have as great of an increase in translation compared to 1079 cells,but this may be due to cell line variations. Mock transfected cells,those treated with transfection reagent only, show no luciferaseactivity in both cell lines.

Introduction of B18R mRNA Prior to Transfection of Luciferase mRNAIncreases Luciferase Activity in Cells.

Transfection of B18R mRNA 10, 24, 36, and 48 hours prior to luciferasemRNA transfection increased luciferase activity (FIG. 4). The folddifference between B18R mRNA-treated and un-treated cells was similar atall treatment times, ranging from 1.7- to 2.2-fold, but greatest at the10-hour treatment time, 2.2-fold change in luciferase activity. Therewas not an advantage to increasing the time between the B18R mRNA andluciferase mRNA transfections to boost luciferase activity.

Introduction of B18R mRNA into Cells Inhibits Type I but not Type IIInterferon Activity.

IFNα, IFNβ and IFNγ have all been shown previously to activate Jak/Statsignaling cascades, ultimately resulting in Interferon Response Factor(IRF) binding to Interferon Stimulated Response Elements (ISREs)eliciting interferon responsive transcriptional activation (Nelson etal., 1993). B18R protein has previously been shown to bind to andinhibit type I interferons (IFNα and IFNβ), but not type II interferons(IFNγ) (Symons et al., 1995). Similarly, based on assays using a Helacell line that contains ISREs linked to the luc2 gene, B18R mRNA madewith ΨTP substituted for UTP (and/or with m⁵CTP substituted for CTP)results in inhibition IFNα and IFNβ activity, but has no effect on IFNγactivity, whereas other Exogenous mRNAs (e.g., EGFP mRNA as a negativecontrol for Agent mRNA comprising B18R mRNA) does not detectably inhibitthe activity of any of the interferons (FIG. 5).

Introduction of E3L or K3L mRNA Prior to Transfection of AlkalinePhosphatase mRNA Increased Alkaline Phosphatase Activity in Cells.

The vaccinia virus E3L and K3L intracellular proteins have been shown toinhibit innate immune system activation elicited by the introduction ofdsRNA into the cytoplasm (Carroll et al., 1993; Chang et al., 1992;Davies et al., 1992; Rice et al., 2011; Xiang et al., 2002). Inhibitionof the innate immune system by expression of E3L or K3L proteinsincreases transcription activation by blocking interferon inductionthrough IRF3, the 2-5A/RNaseL pathway, and the PKR pathway (Carroll etal., 1993; Rice et al., 2011; Xiang et al., 2002). Transfection of ALKPmRNA alone or along with EGFP mRNA as a negative control for Agent mRNAcomprising E3L or K3L mRNA results in similar ALKP reporter activity inC3H10T1/2 mouse mesenchymal stem cells (FIG. 6A) and 1079 humanfibroblasts (FIG. 6B), whereas transfection of ALKP mRNA along witheither E3L or K3L mRNAs resulted in significant increases in ALKPreporter activity compared to ALKP mRNA alone. Transfecting both E3LmRNA and K3L mRNA together with ALKP mRNA resulted in an even higherALKP reporter activity than the affect observed when adding either E3LmRNA or K3L mRNA alone. It is interesting to note that co-transfectionof E3L and/or K3L mRNAs with a reporter mRNA (e.g., ALKP mRNA) issufficient to enhance reporter mRNA translation and activity (FIG. 6).This is not the case with B18R mRNA, which needs to be transfected hoursbefore the reporter mRNA (e.g., luc2 mRNA) is transfected in order toenhance reporter mRNA activity (FIG. 4). E3L and K3L are bothintracellular proteins, while B18R is a secreted protein. The delayedeffect of B18R mRNA in reducing an innate immune response may be due tothe time needed for B18R mRNA to be translated into protein,post-translationally modified, and secreted into the extracellularenvironment, where it exerts its effect. As shown in FIG. 1, B18Rprotein can be added directly to media of cells in culture to enhancetranslation and activity of transfected reporter mRNAs like luciferasemRNA. This shows that secreted proteins that act in the extracellularenvironment have utility for in vitro cell culture, since these proteinsdo not need to introduced or internalized into the cells. Intracellularproteins, like E3L and K3L, could be made and purified as recombinantproteins, but the difficulties of introducing such proteins into cellsis a major obstacle thereby limiting their utility. Potentially, avariant nucleic acid sequence of an intracellular protein, like E3L orK3L, could be generated using methods disclosed herein so that theprotein will have a signal peptide or amino acid sequence which resultsin uptake or internal localization of the protein into the cell, butmaking such a variant is also not easy or reliable. However, due to thedifficulties of delivering intracellular proteins like the E3L or K3Lproteins into cells, the use of an Agent mRNA comprising mRNA thatencodes one or both of the E3L and K3L proteins provides clear benefitsover the proteins themselves for inhibiting an innate immune responseinduced in cells by a Foreign Substance (e.g., a LPS, dsRNA or ExogenousRNA).

Introduction of E3L mRNA Inhibits dsRNA-Induced Interferon Stimulation.

Transfection of dsRNA or in vitro transcribed RNA containing unwanteddsRNA contamination is known to bind to Toll-like receptor 3 (TLR3) andactivate the immune system resulting in interferon production(Alexopoulou et al., 2001; Kariko et al., 2004). Transfection of dsRNA(e.g., LIN28 dsRNA) into the stable Hela cell line expressing InterferonStimulated Response Elements (ISRE) driving luciferase 2 expression(ISRE-luc2) resulted in enhanced luciferase reporter activity as areadout of interferon pathway activation (FIG. 7). Transfections ofLIN28 dsRNA along with c-MYC mRNA as a negative control for Agent mRNAcomprising E3L mRNA does not alter the interferon activation compared toLIN28 dsRNA transfections alone. Transfecting E3L mRNA together with theLIN28 dsRNA substantially reduced the interferon activation compared totransfecting with LIN28 dsRNA alone or co-transfecting with LIN28 dsRNAand c-MYC mRNA.

Introduction of E3L or K3L mRNA into Mesenchymal Stem Cells Facilitates

Their Reprogramming to Myoblasts by MYOD mRNA.

Mouse mesenchymal stem cells can be induced to form muscle myoblasts byoverexpression of the master regulatory transcription factor, MYOD(Davis et al., 1987). Myoblast induction results in the formation ofmultinucleated myoblasts expressing myosin heavy chain (MHC) as a markerof muscle differentiation (Davis et al., 1987). We found that MYOD mRNAcontaining canonical nucleosides (GAUC) could not reprogram C3H10T1/2mesenchymal stem cells into myoblasts after two successive transfections(FIG. 8.C), including wherein the cells were co-transfected with bothMYOD mRNA and EGFP mRNA as a control for Agent mRNA comprising E3L mRNAor K3L mRNA (FIG. 8.D). However, myoblasts were induced when theC3H10T1/2 mesenchymal stem cells were co-transfected with MYOD mRNA(GAUC) together with: E3L mRNA (GAΨm⁵C) (FIG. 8.E), K3L mRNA (GAΨm⁵C)(FIG. 8.F), or both E3L mRNA and K3L mRNA (FIG. 8.G). Co-transfection ofMYOD mRNA together with both E3L mRNA and K3L mRNA did not seem toincrease reprogramming (based on MHC expression) compared toco-transfection of MYOD mRNA with either E3L mRNA or K3L mRNA alone(FIG. 8.G versus FIG. 8.E or 8.F). As expected, no myoblasts wereinduced without MYOD mRNA, as shown for the untreated (FIG. 8.A) andmock-transfected (FIG. 8.B) C3H10T1/2 cells.

Introduction of E3L or K3L mRNA into Human or Mouse Fibroblasts orKeratinocytes Facilitates their Reprogramming to iPS Cells

Human or mouse fibroblasts or keratinocytes that are transfected dailyfor 18 days (at a total daily combined dose of 0.6-1.2 μg (preferably1-1.2 μg) for all Exogenous mRNA (comprising a 3:1:1:1:1:1 molar ratioof GAψC mRNAs encoding OCT3/4, SOX2, KLF4, NANOG, LIN28 and one proteinselected from c-MYC(T58A), c-MYC and L-MYC) per approximately 10⁵ cells)in the presence of E3L GAΨm⁵C mRNA (5 μg/ml), K3L GAΨm⁵C mRNA (5 μg/ml),or both E3L GAΨm⁵C mRNA and K3L GAΨm⁵C mRNA (5 μg/ml of each) complexedwith RNAiMax transfection reagent would result in induction of a highernumber of iPS cells compared to the same cells that are similarlytransfected with the same Exogenous mRNA in the absent of the AgentmRNA. Thus, Agent mRNA comprising E3L GAΨm⁵C mRNA and/or K3L GAΨm⁵C mRNAcan increase iPSC induction. Without being bound by theory, it isbelieved that this increased level of iPSC induction is due to areduction in the innate immune response and/or an increase in thetranslation of the Exogenous mRNAs during the reprogramming period.

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All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

We claim:
 1. A method for reducing an innate immune response in human oranimal somatic cells to reprogram the somatic cells to inducedpluripotent stem cells (iPSCs) comprising: a) introducing into human oranimal somatic cells: i) Agent mRNA comprising in vitro-synthesized GAUCmRNAs or GAΨC mRNAs or GAΨm⁵C mRNAs encoding one, two or all three ofthe Vaccinia virus proteins B18R protein, E3Lprotein, and K3L protein,ii) Exogenous mRNA comprising either GAUC mRNAs or GAΨC mRNAs or GAΨm⁵CmRNAs encoding: i) iPSC reprogramming proteins OCT3/4, SOX2, and KLF4,and ii) one MYC protein selected from c-MYC(T58A), c-MYC and L- MYC and;b) repeating said introducing of said Agent mRNA and said Exogenous mRNAinto said cells and culturing said cells over multiple days, wherein: i)any innate immune response comprising elevated type 1 IFN production orresponse in said cells is reduced compared to the innate immune responsein the absence of introducing said Agent mRNA, ii) the rate of survivalof said cells is increased, and iii) at least some of said somatic cellsare reprogrammed to iPSCs.
 2. The method of claim 1, wherein expressionof said Agent mRNA in said somatic cells: a) increases the translationof said Exogenous RNA in said cells; and/or b) decreases toxicity tosaid cells; and/or c) increases the rate of survival of the cells. 3.The method of claim 1, wherein said Agent mRNA further comprises GAUCmRNA or GAΨC mRNA or GAΨm5C mRNA that encodes a dominant negativefunctional inhibitor of TP53 protein.
 4. The method of claim 1, whereinsaid introducing of said Agent mRNA occurs up to about 24 hours prior tosaid introducing of said Exogenous mRNA.
 5. The method of claim 1,wherein said introducing of said Agent mRNA occurs together with or atapproximately the same time as said Exogenous mRNA.
 6. The method ofclaim 1, wherein said Exogenous mRNA and/or said Agent mRNA is invitro-transcribed (IVT) RNA comprising (i) a cap that was added to their5′-termini co-transcriptionally by incorporation of a cap analog duringin vitro transcription or post-transcriptionally using capping enzymesthat results in a cap with a cap0 structure or with a cap1 structure,and (ii) a poly(A) tail that was added to their 3′-terminico-transcriptionally using a DNA template that encodes the poly(A) tail,or post-transcriptionally using a poly(A) polymerase.
 7. The method ofclaim 1, wherein said Exogenous mRNA and/or Agent mRNA comprise onlyGAUC mRNAs made by in vitro transcription (IVT) in the presence of thecanonical unmodified ribonucleoside-5′-triphosphates GTP, ATP, UTP, andCTP, and a cap analog if said GAUC mRNAs are cappedco-transcriptionally.
 8. The method of claim 1, wherein said ExogenousmRNA and/or said Agent mRNA comprise mRNAs that contain pseudouridine(Ψ) in place of uridine and/or 5-methylcytidine (m⁵C) in place ofcytidine, which mRNAs are made by IVT in the presence of ΨTP in place ofUTP, and/or m⁵CTP in place of CTP, and a cap analog if said GAΨC,GAΨm⁵C, or GAUm⁵C mRNAs are capped co-transcriptionally.
 9. The methodof claim 1, wherein said Agent mRNA further comprises GAUC mRNA or GAΨCmRNA or GAΨm⁵C mRNA that encodes SV40 Large-T antigen.
 10. The method ofclaim 1, wherein said Exogenous mRNA further comprises GAUC mRNA or GAΨCmRNA or GAΨm⁵C mRNA that encodes NANOG and/or LIN28.
 11. The method ofclaim 10, wherein said mRNAs of said Exogenous mRNA or said Agent mRNAcomprise: (i) a cap that was added to their 5′-terminico-transcriptionally by incorporation of a cap analog during in vitrotranscription or post-transcriptionally using a capping enzyme thatresults in a cap with a cap0 structure or with a cap1 structure, and(ii) a poly(A) tail that was added to their 3′-terminico-transcriptionally using a DNA that encodes the poly(A) tail orpost-transcriptionally using a poly(A) polymerase.
 12. The method ofclaim 10, wherein said Exogenous mRNA and/or Agent mRNA comprise onlyGAUC mRNAs made by in vitro transcription (IVT) in the presence of onlythe canonical unmodified ribonucleoside-5′-triphosphates GTP, ATP, UTP,and CTP, and a cap analog if said GAUC mRNAs are cappedco-transcriptionally.
 13. The method of claim 10, wherein said ExogenousmRNA and/or said Agent mRNA comprise mRNAs that contain pseudouridine(Ψ) in place of uridine and/or 5-methylcytidine (m⁵C) in place ofcytidine, which mRNAs are made by IVT in the presence of only themodified ribonucleoside-5′-triphosphates GTP, ATP, ΨTP in place of UTP,and/or m⁵CTP in place of CTP, and a cap analog if said GAΨC, GAΨm⁵C, orGAUm⁵C mRNAs are capped co-transcriptionally.
 14. The method of claim 1,wherein said introducing of said Agent mRNA occurs up to about 24 hoursprior to said introducing of said Exogenous mRNA.
 15. The method ofclaim 1, wherein said introducing of said Agent mRNA occurs togetherwith or at approximately the same time as said Exogenous mRNA.
 16. Themethod of claim 1, wherein said Agent mRNA is purified so that less than0.01% of the total mass or weight of RNA comprising said Agent mRNAconsists of double-stranded RNA (dsRNA) of a size greater than about40-basepairs in length when assayed by dot blot immunoassay using the J2dsRNA-specific monoclonal antibody.
 17. The method of claim 1, whereinsaid Agent mRNA further comprises GAUC mRNA or GAΨC mRNA or GAΨm⁵C mRNAthat encodes a dominant negative functional inhibitor of TP53 protein.18. The method of claim 1, wherein the method further comprises, priorto step a), the step of contacting said somatic cells with an effectiveamount of a protein that is capable of reducing an innate immuneresponse comprising elevated type 1 IFN production or response.
 19. Themethod of claim 1, wherein said Exogenous mRNA is purified so that lessthan 0.01% of the total mass or weight of RNA comprising said ExogenousmRNA consists of double-stranded RNA (dsRNA) of a size greater thanabout 40-basepairs in length when assayed by dot blot immunoassay usingthe J2 dsRNA-specific monoclonal antibody.